Peptide ligands of the urokinase receptor

ABSTRACT

Novel peptides that are capable of binding to uPAR and inhibiting the binding of an integrin and vitronectin are described. Also provided are nucleic acid sequences encoding the novel peptides. Methods for screening for small molecules, other peptides, or peptoids that mimic the antagonistic function of the peptides of the invention are described. The invention has applications in design of therapeutics for treating disorders characterized by upregulation of uPA and uPAR, and cancer and chronic inflammation, cell migration or uPAR: integrin binding interactions, and diagnostical applications to such disorders.

FIELD OF THE INVENTION

[0001] This invention relates to the identification of novel functionalsites on the urokinase receptor in the presence of the receptor bindingregion of urokinase. Described herein are peptides derived frombacteriophage display that identify the sites, and a general method foridentifying functional sites on proteins using bacteriophage display.Also, methods of using urokinase receptor functional sites for studiesof vitronectin and integrin interaction with urokinase:urokinasereceptor complex interaction are described. Also described are uses ofthe instant peptides for developing therapeutic molecules capable ofantagonisting interactions of the vitronectin and integrin peptides withthe urokinase:urokinase receptor complex.

BACKGROUND OF THE INVENTION

[0002] The urokinase plasminogen activator (uPA) is a serine proteasethat interacts with its cell surface receptor (uPAR) providing aninducible, localized cell surface proteolytic activity, therebypromoting cellular invasion. The uPA:uPAR complex converts plasminogeninto plasmin which is known to degrade various matrix glycoproteins asdescribed in Ellis et al, J. Biol. Chem. 264: 2185-2188 (1989), Vassiliet al, J. Clin. Invest. 88: 1067-1072 (1991), and Mignatti and Rifkin,Physiol. Rev. 73: 161-195 (1993). The simaltaneous expression of uPA andits receptor has been associated with localized plasminogen activationand pericellular matrix degradation during directed cell migration ofnormal and tumor cells.

[0003] The urokinase receptor (uPAR) is a 283 amino acidglycosylphosphatidyl-inositol (GPI)—anchored receptor protein ofurokinase and vitronectin which appears to be a triplication of a 90amino acid domain as described in Plough, and Ellis, FEBS Lett.349:163-168 (1994) and Roldan et al, EMBO J. 9: 467-474 (1990).Proteolysis of uPAR can yield fragments composed of domain 1 and domains2-3, and subsequent analysis has shown that disulfide bonding pattern ofdomain 1 is completely internal to the domain, as described in Plough etal, J.Biol.Chem. 268:17539-17546 (1993), and Kieffer et al, Biochem.33:4471-4482 (1994).

[0004] The migration and invasion of cells appear to require cellsurface localized proteolysis and adhesion to specific components of theextracellular matrix. These processes are necessary for many normal andpathological processes, including tissue remodeling, embryoimplantation, angiogenesis, and tumor cell invasion and metastasis asdescribed in Fazioli et al, Trends Pharimacol.Sci. 15:25-29(1994), andMignatti et al, Physiol.Rev. 73:161-195 (1993). Important components ofthe cell surface proteolytic and cellular adhesion cascades are theplasminogen activator/plasmin system, matrix metalloproteinases, andintegrins, as described in Felding-Habermann et al, Curr.Biol. 5 864-868(1993). Adhesion to the extracellular matrix component vitronectin hasbeen reported to correlate with UPAR expression, and uPA binding sitesand vitronectin receptors have been shown to colocalize on HT1080 cells,as described in Waltz et al, J.Biol.Chem. 269: 14746-14750 (1994)., andCiambrone et al, J.Biol.Chem. 267: 13617-13622 (1992). More recently ithas been demonstrated that uPAR can function as a cell adhesion receptorfor vitronectin in a uPA dependent manner as described in Wei et al,J.Biol.Chem. 269: 32380-32388 (1994).

[0005] Early experiments using chemical cross-linking suggested that thefirst domain of uPAR was sufficient for high affinity binding of uPA,however, subsequent work has shown that an intact 3-domain molecule isrequired, and that additional binding determinants in domains 2 and 3are likely involved, as described in Plough et al, Biochem. 3: 8991-8997(1994). The undefined interactions may be with the uPA EGF-like domainor indirect interactions affecting the conformation of domain 1.Previous work has been unsuccessful in distinguishing whether domain 2and 3 has measurable affinity for uPA, because of the difficulty ofseparating domain 2 and 3 from trace amounts of full length uPAR asdescribed in Plough et al, Biochem. 3: 8991-8997 (1994).

[0006] The uPA:uPAR system has been identified as promoting pericellularproteolysis, and functions attributable to uPAR include cell migration,adhesion and mitogenesis. It would be desirable, therefore, to elucidatethe function of domains 2 and 3 of uPAR.

SUMMARY OF THE INVENTION

[0007] A first embodiment of the invention is a method of identifying anorphan binding site on a target polypeptide sequence by

[0008] (a) providing

[0009] (1) a library of potential ligands,

[0010] (2) a target polypeptide in contact with a known ligand for thetarget polypeptide,

[0011] (b) contacting the target polypeptide and known ligand with thelibrary of potential ligands, and

[0012] (c) identifying the potential ligand that binds to the targetpolypeptide in the presence of the known ligand to form a binding pairwith the target polypeptide and known ligand.

[0013] Another embodiment of the invention is an isolated peptide thatbinds a urokinase plasminogen activator receptor (uPAR) and inhibitsuPAR binding to an integrin. The isolated peptide can be YHXLXXYMYT(SEQU ID NO:5) or AESTYHHLSLGYMYTLN (SEQ ID NO:4).

[0014] Another embodiment of the invention is an isolated peptide thatbinds a urokinase plasminogen activator (uPAR) and inhibits uPAR bindingto vitronectin. The isolated peptide can be AEPVYQYELDSYLRSYY (SEQ IDNO: 1), AEFFKLGPNGYVYLHSA (SEQ ID NO:2), or AELDLSTFYDIQYLLRT (SEQ IDNO:3) or FKLXXXGYVYL (SEQ ID NO:6).

[0015] Yet another embodiment of the invention is an isolated nucleicacid sequence that encodes a peptide that binds a urokinase plasminogenactivator receptor (uPAR) and inhibits uPAR binding to an integrin. Theisolated nucleic acid sequence can encode the amino acid sequence ofYHXLXXGYMYT (SEQ ID NO:5) or STYHHLSLGYMYTLN (SEQ ID NO:4).

[0016] Still another embodiment of the invention is an isolated nucleicacid sequence that encodes a peptide that binds a urokinase plasminogenactivator receptor (uPAR) and inhibits uPAR binding to vitronectin. Theisolated nucleic acid sequence can encode the amino acid sequence ofAEPVYQYELDSYLRSYY (SEQ ID NO:1), or FFKLGPNGYVYLHSA (SEQ ID NO:2) or,AELDLSIFYDIQYLLRT (SEQ ID NO:3) or FKLXXXGYVYL (SEQ ID NO:6).

[0017] Yet another embodiment of the invention is a method of treating apatient with a disorder characterized by upregulation of uPA and uPAR byproviding an effective amount of an antagonist of a uPAR:integrinbinding pair, and administering the antagonist to the patient.

[0018] An additional embodiment of the invention is a method ofscreening for an antagonist of uPAR:integrin interaction comprising thesteps of providing a peptide antagonist of a uPAR:integrin interaction,competing the peptide antagonist with a candidate antagonist for bindingto uPAR, and identifying a candidate antagonist by the ability tocompete with the peptide antagonist for uPAR binding.

[0019] Still a further embodiment of the invention is a small moleculeantagonist of a uPAR:integrin interaction identified by the justdescribed method; a peptide antagonist of a uPAR:integrin interactionidentified by that method; and a peptoid antagonist of a uPAR:integrininteraction identified by the same method.

[0020] Another embodiment of the invention is a pharmaceuticalcomposition for treating a disorder characterized by upregulation of uPAand uPAR comprising an effective amount of an antagonist of auPAR:integrin binding pair and a pharmaceutically acceptable carrier.

[0021] Yet another embodiment of the invention is a pharmaceuticalcomposition for treating a patient with a disorder characterized byupregulation of uPA and uPAR comprising an effective amount of a nucleicacid encoding a peptide antagonist of a uPAR:integrin binding pair and apharmaceutically acceptable carrier suitable for expressing the peptidein the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1. UPA1-48 is required for sUPAR binding to vitronectin.Various concentrations of uPA1-48 were incubated with biotinylated sUPARin vitronectin-coated wells and vitronectin-bound sUPAR detected asdescribed in the examples. Each determination was in duplicate and theresults are reported as the mean absorbance at 450 nm of the sUPAR plusuPA1-48 samples minus the mean absorbance of the sUPAR alone sample(approximately 0.03).

[0023]FIG. 2. Effects of various peptide ligands on sUPAR binding tovitronectin. The effects of the indicated peptides on sUPAR/vitronectininteraction were determined by incubating the peptides with biotinylatedsUPAR in vitronectin-coated wells in the presence of uPA1-48 as inFIG. 1. All peptides were solubilized in 100% DMSO before diluting tothe indicated concentrations with PBS/2% BSA for the assay. Controlsamples included suPAR plus 20 nM UPA1-48 and sUPAR alone. Peptidestested were clone 7, clone 7S (scrambled clone 7), clone 18, clone 25,clone 20, clone 20A (L to A replacement at position 14), and uPA 13-32C19A. Results are reported as the mean OD₄₅₀ values of triplicatepoints. Where error bars are not shown they are smaller than thesymbols.

[0024]FIG. 3. Peptides 7 and 18 are Homologous to the Somatomedin BDomain of Vitronectin. The sequence of vitronectin from residues 1-47including the somatomedin B domain and RGD motif is compared with thesequences of clones 7 and 18. Homologous residues at positions 22-28 invitronectin and in the bacteriophage derived peptides are in bold as isthe RGD sequence in vitronectin.

[0025]FIG. 4. Alanine Replacement of Peptide 7 Affects BothBacteriophage and Vitronectin Binding to UPAR. Synthetic peptides at 40μM were tested as competitors for binding of bacteriophage 7 tobiotinylated suPAR as described in Materials and Methods and shown inpanel A. Bacteriophage were detected with a rabbit anti-M13 antibody asdescribed. The indicated values are the mean of triplicatedeterminations. The same peptides were tested in triplicate at 50 μM inthe uPA1-48:uPAR:vitronectin binding ELISA.

[0026]FIG. 5. Bacteriophage binding to sUPAR domain 2/3. Phage wereadded to wells containing sUPAR domain 2/3 immobilized by its epitopetag via protein G and monoclonal antibody to the epitope tag. Wellscontaining protein G and antibody but no domain 2/3 were included todetermine nonspecific phage binding. Urea-eluted phage and the inputstocks were titered by plaque formation assay. Results were single pointdeterminations calculated as a percent of the input titer and wererepeated in three separate experiments.

[0027]FIG. 6: Table. The table of FIG. 6 depicts the sequences, phageyields, and IC50s in uPAR binding assays for selected phage peptides.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] The invention described herein draws on previously published workand pending patent applications. By way of example, such work consistsof scientific papers, patents or pending patent applications. All suchpublished work cited herein are hereby incorporated by reference. Theinvention can be better understood in light of the following definitionsincorporated herein.

[0029] Definitions

[0030] The term “orphan binding site” as used herein refers to apreviously unidentified site on a polypeptide sequence that is capableof binding to another peptide or polypeptide sequence. The orphanbinding site is distinguishable from a binding site for which the nativeligand is known. The orphan binding sites of the invention arediscovered by phage display of a peptide sequence that is capable ofbinding a site on a target polypeptide. The binding site may involvebinding of a third or fourth additional polypeptide, for example, wherethe urokinase plasminogen activator receptor (uPAR) binds urokinaseplasminogen activator (uPA) in addition to binding other ligands orpolypeptides, such as, for example vitronectin and integrin.

[0031] The term “orphan polypeptide” as used herein refers to apolypeptide sequence capable of binding at an orphan binding site. Theorphan polypeptide may be, for example, a peptide used in a phagedisplay screening to determine orphan binding sites, or may be thepolypeptide sequence of a native or synthetic molecule that binds theorphan binding site, and is homologous in sequence to the peptide usedto determine the location of the orphan binding site.

[0032] The term “potential ligand” as used herein refers to any peptide,polynucleotide, polysaccharide, or other molecule that could potentiallybind to the target polypeptide.

[0033] The term “potential ligand library” as used herein refers to acollection or mixture of at least 50 compounds that are potentialligands as defined above, and more preferably a potential ligand libraryis at least 200 potential ligand compounds, and still more preferablymore than 500 compounds.

[0034] The term “unknown ligand” as used herein refers to ligands of atarget polypeptide that have not yet been discovered, but that may bediscovered by the method of the invention. Where a potential ligand canbind a target polypeptide, and antagonize binding of a previouslyunknown ligand, the identity and existence of the unknown ligand can bedetermined either by structural analysis of the potential ligand thatbinds a target polypeptide, or by functional changes that indicate thatbinding has been disrupted by an antagonist. The unknown ligand can alsobe determined by screening a library of polypeptides comprisingsequences that occur naturally in a competition assay with the potentialligand bound to the target polypeptide at the orphan binding site.

[0035] The term “bacteriophage library” as used herein refers to thetechnique in molecular biology of creating a library of peptidesexpressed on the surface of a bacteriophage for presentation andcontacting potential target polypeptides. The library is thepolynucleotides that are expressable as peptides and presented by thebacteriophage, and may be the DNA or the amino acid moieties used orgenerated by this technique. Bacteriophage panning or display hasapplications as described herein for screening for ligands of targetpolypeptides, which when identified, also identifies orphan bindingsites on the target polypeptides.

[0036] The term “peptide” and the term “polypeptide” as used hereinrefers to a peptide or a polypeptide produced in vivo or in vitro in anenvirornent manipulated by humans using techniques of molecular biology,biochemistry or gene therapy. For example, an isolated peptide orpolypeptide can be produced in a cell free system by automated peptideor polypeptide synthesis, in heterologous host cells transformed withthe nucleic acid sequence encoding the peptide or polypeptide andregulatory sequences for expression in the host cells, and in an animalinto which the coding sequence of the peptide or polypeptide has beenintroduced for expression in the animal. A peptide or polypeptideisolated for purposes herein to the extent that it is not present in itsnatural. state inside a cell as a product of nature. For example, suchisolated polypeptides or polynucleotides can be 10% pure, 20% pure, or ahigher degree of purity.

[0037] The term “derivative” as used herein in reference to a peptide,polypeptide or a polynucleotide means a peptide, polypeptide orpolynucleotide that retains the functionality of the peptide,polypeptide or polynucleotide to which it is a derivative. They may bevariously modified by amino acid deletions, substitutions, insertions orinversions by, for example, site directed mutagenesis of the underlyingnucleic acid molecules. Derivatives of a peptide, polypeptide orpolynucleotide may also be fragments thereof. In any case, a derivative,or a fragment, retains at least some, and preferably all of the functionof the peptide or polypeptide from which it is derived.

[0038] The term “pharmaceutical composition” refers to a composition foradministration of a therapeutic agent. The therapeutic agent can be, forexample a peptide, a polypeptide, a polynucleotide, a small molecule, apeptoid, or a derivative of any of these, and refers to anypharmaceutical carrier that does not itself induce the production ofantibodies harmful to the individual receiving the composition, andwhich may be administered without undue toxicity.

[0039] Administration of a therapeutic agent of the invention includesadministration of a therapeutically effective amount of the agent of theinvention. The term “therapeutically effective amount” as used hereinrefers to an amount of a therapeutic agent sufficient to treat orprevent a condition treatable by administration of a composition of theinvention. That amount is the amount sufficient to exhibit a detectabletherapeutic, preventitive or ameliorative effect. The effect mayinclude, for example, treatment or prevention of the conditions listedherein. The precise effective amount for a subject will depend upon thesubject's size and health, the nature and extent of the condition beingtreated, recommendations of the treating physician, and the therapeuticsor combination of therapeutics selected for administration. Thus, it isnot useful to specify an exact effective amount in advance. However, theeffective amount for a given situation can be determined by routineexperimentation. Administration can include admininistration of apolypeptide, and causing the polypeptide to be expressed in an animal byadministration of a polynucleotide encoding the polypeptide.

[0040] A “recombinant vector” herein refers to any vector for transferor expression of the polynucleotides herein in a cell, including, forexample, viral vectors, non-viral vectors, plasmid vectors and vectorsderived from the regulatory sequences of heterologous hosts andexpression systems.

[0041] A “regulatory sequence” herein refers to a nucleic acid sequenceencoding one or more elements that are capable of affecting or effectingexpression of a gene sequence, including transcription or translationthereof, when the gene sequence is placed in such a position as tosubject it to the control thereof. Such a regulatory sequence can be,for example, a minimal promoter sequence, a complete promoter sequence,an induced active promoter, an enhancer sequence, an upstream activationsequence (“UAS”), an operator sequence, a downstream terminationsequence, a polyadenylation sequence, an optimal 5′ leader sequence tooptimize initiation of translation, or a Shine-Dalgarno sequence.Alternatively, the regulatory sequence can contain a hybrid of promotersof any of the above, such as a hybrid enhancer/promoter element. Theregulatory sequence that is appropriate for expression of the gene ofinterest differs depending upon the host system in which the constructis to be expressed. Selection of the appropriate regulatory sequencesfor use herein is within the capability of one skilled in the art. Ineukaryotes, for example, such a sequence can include one or more of apromoter sequence and/or a transcription termination sequence.Regulatory sequences suitable for use herein may be derived from anysource including a prokaryotic source, an eukaryotic source, a virus, aviral vector, a bacteriophage or a linear or circular plasmid. Theregulatory sequence herein can also be a synthetic sequence, forexample, one made by combining the UAS of one gene with the remainder ofa requisite promoter from another gene, such as the GADP/ADH2 hybridpromoter. A regulatory sequence can also be a repressor sequence.

[0042] “Mammalian cell” as used herein refers to a subset of eukaryoticcells useful in the invention as host cells, and includes human cells,and animal cells such as those from dogs, cats, cattle, horses, rabbits,mice, goats, pigs, etc. The cells used can be genetically unaltered orcan be genetically altered, for example, by transformation withappropriate expression vectors, marker genes, and the like. Mammaliancells suitable for the method of the invention are any mammalian cellcapable of expressing the genes of interest, or any mammalian cells thatcan express a cDNA library, cRNA library, genomic DNA library or anyprotein or polypeptide useful in the method of the invention. Mammaliancells also include cells from cell lines such as those immortalized celllines available from the American Type Culture Collection (ATCC). Suchcell lines include, for example, rat pheochromocytoma cells (PC12cells), embryonal carcinoma cells (P19 cells), Chinese hamster ovary(CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidneycells (COS), human hepatoceflular carcinoma cells (e.g., Hep G2), humanembryonic kidney cells, mouse sertoli cells, canine kidney cells,buffalo rat liver cells, human lung cells, human liver cells, mousemammary tumor cells, as well as others. Also included are hematopoeticstem cells, neuronal stem cells such as neuronal sphere cells, andembryonic stem cells (ES cells).

[0043] A “polynucleotide sequence,” a “nucleic acid molecule,” a“nucleic acid sequence,” or a “coding sequence,” as used herein, refersto either RNA or DNA that encodes a specific amino acid sequence or itscomplementary strand. A nucleic acid molecule may also be anoligonucleotide probe that may or may not encode a functional peptide,for example, an antisense oligonucleotide sequence, or a ribozyme.

[0044] The term “analog” as used herein refers to splice variants,truncations, variants, alleles and derivatives and the like, of a matureprotein. Unless specifically mentioned otherwise, the “analogs” possessone or more of the bioactivities of the “mature protein,” or possess thebioactivity of the peptide. Thus, peptides or polypeptides that areidentical or contain at least 60%, preferably 70%, more preferably 80%,and most preferably 90% amino acid sequence homology to the amino acidsequence of the mature protein or the peptide wherever derived, fromhuman or nonhuman sources, are included within this definition.

[0045] The “variants” herein contain amino acid substitutions,deletions, or insertions. The amino acid substitutions can beconservative amino acid substitutions or substitutions to eliminatenon-essential amino acid residues such as to alter a glycosylation site,a phosphorylation site, an acetylation site, or to minimize misfoldingby substitution or deletion of one or more cysteine residues that arenot necessary for function. Conservative amino acid substitutions arethose that preserve the general charge, hydiophobicity/hydrophilicityand/or steric bulk of the amino acid substituted, for example,substitutions between the members of the following groups areconservative substitutions: Gly/Ala, Val/Ile/Leu, Asp/Glu, Lys/Arg,Asn/Gln, Ser/Thr/Cys and Phe/Trp/Tyr. The analogs herein further includepeptides having one or more peptide mimics, also known as peptoids, thatpossess the bioactivity of the protein. Included within the definitionare also polypeptides containing one or more analog amino acid(including, for example, unnatural amino acids, etc.), polypeptides withsubstituted linkages, as well as other modifications known in the art,both naturally occurring and non-naturally occurring. The termpolypeptide also does not exclude post-expression modifications of thepolypeptide, for example, glycosylations, acetylations, phosphorylationsand the like.

[0046] The term “binding pair” refers to a pair of molecules, usuallyreferring to a protein/protein pair, but does not exclude a protein/DNApair, or a protein/RNA pair, or DNA/DNA pair, DNA/RNA pair or RNA/RNApair, and can include small molecules that bind protein or DNA or RNA.The components of such pair bind specifically to each other with ahigher affinity than to a random molecule, such that upon binding, forexample, in case of a ligand/receptor interaction, the binding pairtriggers a cellular or an intracellular response, or forms a complex. Anexample of a ligand/receptor binding pair is a pair formed betweenPDGF(platelet derived growth factor) and a PDGF receptor. An example ofa different binding pair is an antigen/antibody pair in which theantibody is generated by immunization of a host with the antigen. Anexample of an organic molecule—protein binding pair is the binding ofretinoic acid with its protein receptor, the retinoic acid receptor.Specific binding indicates a binding interaction having a lowdissociation constant, which distinguishes specific binding fromnon-specific, background, binding. A low dissociation constant would be,for example, 1.0 μM, more preferably 10 nM, still more preferably 1.0 nMor less.

[0047] The term “antagonist” as used herein refers to a molecule thatblocks signalling to a detectable degree, as for example, a moleculethat can bind a receptor, but which does not cause a signal to betransduced by the receptor to the cell. In the case of an antagonistpeptide, the peptide antagonist can bind, for example, the uPAR receptorat or near the integrin binding site, and prevent integrin from forminga binding pair with uPAR.

[0048] The term “agonist” as used herein refers to a molecule thatmimics the signalling in the pathway under study, for example, bybinding a receptor and promoting a signal transduction to the cellthrough the receptor. In the case of the invention, an agonist of apeptide antagonist of uPAR would mimic or be able to compete with thepeptide antagonist for blocking the formation of a uPAR:integrin bindingpair. Small molecules or peptoids can be screened for the ability toperform the same or similar function of a peptide antagonist of theuPAR:integrin binding pair interaction.

[0049] The urokinase plasminogen activator receptor “uPAR” as usedherein refers to the urokinase plasminogen activator receptor. uPAR is aglycosylphosphatidyl-inositol-linked urokinase and vitronectin receptor.uPAR is expressed on many cells as a consequence of cytokine stimulationor malignant transformation as described in Blasi et al, J. Cell. Biol.104: 801(1987).

[0050] Urokinase plasminogen activator “uPA” as used herein refers to aserine protease capable of activating urokinase plasminogen. When boundto its cell surface receptor, uPAR, uPA converts plasminogen to plasmin.

[0051] “Integrin” as used herein refers to the integrin farily of celladhesion receptors known to mediate cell attachment to extracellularmatrix proteins and also known to play a critical role in cell motility.

[0052] The term “cytoskeletal disorder” as used herein refers to adisorder in a patient that can be characterized at least in part by theformation of an abnormal condition in the cytoskeleton of at least onetissue of the patient. Cytoskeletal abnormalities can be associated witha variety of conditions, including, for example, tumor growth,metastatic cancer, angiogenesis, wounds, and other disorders.

[0053] Some of the abbreviations used herein are: EGF, epidermal growthfactor; uPA, urokinase plasminogen activator, uPA1-48, amino acids 1 to48 of urokinase; uPAR, urokinase plasminogen activator receptor; sUPAR,soluble truncated form of the urokinase receptor; uPA13-32, amino acids13-32 of human urokinase with Cys19 converted to Ala; PAI-1, plasminogenactivator inhibitor type-1; ATF, amino terminal fragment of uPA; HRP,horse radish peroxidase; PBS, phosphate buffered saline; BSA, bovineserum albumin.

[0054] The invention is the use of bacteriophage display to identifynovel functional sites on proteins. Using this novel application ofbacteriphage display techniques, the inventors have identified novelpeptide sequences that bind to the human urokinase receptor in thepresence of the receptor binding region of human urokinase, and soidentified novel functional sites.

[0055] Accordingly, the identified peptides define two new functionalsites on the urokinase receptor. The first is a site that corresponds tothe interaction site of urokinase:urokinase receptor complexes withvitronectin and show homology to the somatomedin B domain ofvitronectin. The second functional site is involved in a previouslyunexpected interaction of the urokinase receptor with integrins andlikely defines the integrin:urokinase receptor interface. Modulation ofthis second site can lead to alterations in integrinactivity/specificity and affect cell adhesion and other integrinmediated events.

[0056] The invention includes three peptides that inhibit theuPAR:vitronectin binding interaction, peptide 7 (SEQ ID NO:1), peptide 9(SEQ ID NO:2), and peptide 18 (SEQ ID NO:3), and use of these peptidesto inhibit the uPAR:vitronectin interaction. Vitronectin has beenimplicated in binding to uPAR as described in Waltz et al, J.Biol.Chem.269: 14746-14750 (1994). It has been shown that the urokinase receptorcan be a uPA dependent adhesion receptor for vitronectin as described inWei et al, J.Biol.Chem. 269:32380-32388 (1994). Vitronectin is a complexglycoprotein with a modular domain structure which exists in bothcirculating and extracellular matrix forms as described in Preissner etal, Annu.Rev.Cell Biol. 7: 275-310(1991). It interacts with a variety ofcell surface components, including integrins with the alpha-v subunit asdescribed in Felding-Habrann et al, Curr.Biol. 5,864-868. (1993), aswell as with the active conformation of PAI-1 as described in Mimuro etal, J.Biol.Chem. 264: 936-939 (1989). This latter interacton appears tobe via the somatomedin B domain of vitronectin as described in Seiffertet al, J.Biol.Chem. 266: 2824-2830(1991), and Seiffert et al,J.Biol.Chem. 269: 2659-2666 (1984). More recently it has been shown thatvitronectin colocalizes with uPA in the extracular matrix at focalcontacts as described in Ciambrone et al, J.Biol.Chem. 267: 13617-13622(1992). An explanation of this phenomenon was provided by thedemonstration that uPAR is an adhesion rcceptor for vitronectin, whosebinding is stimulated by uPA as described in Wei et al, J.Biol.Chem.269: 32380-32388 (1994).

[0057] We have discovered 15 mer peptides from bacteriophage displaythat inhibit the binding of uPA1-48:uPAR complexes to vitronectin invitro ad tht block the adhesion of U937 cells to vitronectin. Thesepeptides show homology with the somatomedin B domain of vitronectin. Thehomology suggests that the binding sites of uPAR:uPA1-48 complexes andPAI-1 may overlap, which is shown by the fact that PAI-1 competes forbinding of these complexes to vitronectin. The putative alignment of thebacteriophage derived peptides and vitronectin sequence suggests thatbinding of uPAR:uPA1-48 complexes occurs close to the binding site ofa_(v) integrins, as defined by the RGD sequence found at residues 45-47,only 16 amino acids away from the C-terminus of the uPAR binding site.The proximity of these binding sites in vitronectin suggests thepossibility of cooperative interactions between uPAR and integrins. Suchan interaction might provide a mechanism for the signalling capabilityof uPAR via functional coupling with integrin vitronectin receptors,where vitronectin serves to cross-link uPAR and the integrin. This wouldprovide an explanation for how a GPI-linked integral membrane proteintransmits signals to the cell.

[0058] The invention also includes specific peptides that representexamples of a uPAR:integrin site, such as peptide 25 (SEQ ID NO:4).Clone 25 represents a distinct sequence motif, and based on theequivalent binding to D23, identify a unique binding site on suPAR. Thesequence motif we have determined to be necessary but not sufficient forinhibiting the binding pair interaction between uPAR and integrin isGYZY, where Z is M or V. Peptide 25 has been shown to bind to theurokinase receptor and modulates integrin function. The sequence ofpeptide 25 is AESTYHHLSLGYMYTLN, where, by alanine replacement the aminoacids YHXLXXGYMYT, where X is any amino acid were determined to beimportant for inhibiting uPAR binding to integrin.

[0059] A further aspect of the invention is the use of peptide 25 as alead compound and a tool for assay development of other molecules withthe same activity, for example, small molecules and peptoids.

[0060] Other workers have shown that uPAR and both b₂ integrins,specifically Mac-1, and a_(v)b₃ and a_(v)b₅ appear to colocalize incells as described in Xue et al. J. Immunol. 152: 4630-4640 (1994),Bohuslav et al, J. Exp. Med. 181: 1381-1390 (1995), Conforti et al,Blood 83: 994-1005 (1994), and Reinartz et al, Exp. Cell Res. 220:271-282 (1995). However, in none of these cases was there a direct probefor looking at the potential biochemical interaction between uPAR andthe integrins.

[0061] Previous work had demonstrated that selection of high affinitypeptide ligands for the uPA binding site on uPAR was a relativelyefficient process, as described in Goodson et al, Proc.Natl.Acad.Sci.USA 91: 7129-7133 (1994). We extended this analysis by selecting forpeptide-displaying bacteriophage with affinity for additional,functionally important sites on uPAR by including an excess ofrecombinant EGF-like domain of uPA (uPA1-48) to reduce selection of uPAbinding site peptides, as described in Stratton-Thomas et al. Prot.Eng.8: 463470 (1995). The EGF-like domain is the receptor binding motif andbinds to uPAR with similar affinity (0.1-5 nM) as uPA. The 15 mer randompeptide bacteriophage library, as described in Devlin et al, Science249: 404-406 (1990) was affinity selected on suPAR:uPA1-48 complexesimmobilized on magnetic beads.

[0062] In order to analyze the effects of the various peptide ligands onthe uPAR:vitronectin interaction, we developed an in vitro ELISA basedassay for this interaction. Under the conditions of the assay binding ofbiotinylated uPAR to vitronectin is strictly dependent on uPA1-48, asshown in FIG. 1. The apparently stoichiometric binding of theuPA1-48:suPAR complexes to vitronectin indicates that the affinity ofthis interaction is higher than the concentration of complex (Kd<20 nM).

[0063] The ability of the various bacteriophage derived peptides toaffect binding of uPA1-48:uPAR complexes to vitronectin was thenassessed in the ELISA assay. Two classes of peptides were effectiveantagonists in this assay. First, clone 20 and uPA13-32, which competedirectly for uPA1-48 binding to sUPAR, reduce binding. An analog ofclone 20 peptide, which shows greatly reduced receptor binding activity,did not affect binding to vitronectin. Second, clones 7 and 18, whichshow greatly reduced competition for uPA1-48 binding (see table in FIG.6) also inhibit complex binding, while a scrambled version of clone 7(having the same amino acids as clone 7, but in a different order) doesnot. None of the peptides when tested alone increased the binding ofbiotinylated sUPAR to vitronectin.

[0064] A third peptide, clone 25, bound efficiently to suPAk as abacteriophage, had little or no effect on uPA1-48 stimulated vitronectinbinding.

[0065] In order to test whether the clone 7 and 18 peptides bounddirectly at the vitronectin binding site on uPAR, and inhibitedvitronectin binding by uPAR:uPA1-18 by direct competition for that site,the inventors examined the effects of vitronectin on the binding ofthese bacteriophage. Vitronectin reduced bacteriophage binding to theuPA1-48: suPAR complex by 5-10 fold, consistent with the hypothesis thatthese peptides mirnic vitronectin as a uPAR ligand.

[0066] Previous results had shown that vitronectin binding by uPARcorrelated with cell adhesion of stimulated U937 cells as described inWei etal, J.Biol. Chem. 269: 32380-32388 (1994). Whether clone 7 peptidecould block uPAR mediated adhesion of these cells was then tested, withthe result that clone 7 is an effective blocker of uPAR:vitronectininteraction, whereas a scrambled version of the same peptide showed noeffect.

[0067] We demonstrated that binding of uPA1-48:uPAR complexes tovitronectin is blocked by PAI-1, vitronectin, and the somatomedin Bdomain of vitronectin. Another function of vitronectin is to stabilizethe active conformation of PAI-1, which appears to occur via thesomatomedin B domain of vitronectin as described in Seiffert et al,J.Biol.Clzem. 269: 2659-2666 (1994). PAI-1 is a very efficientcompetitor of uPA1-48:suPAR complexes binding to vitronectin, with anapparent IC50 of 10 nM. This suggested that the binding site of uPAR andPAI-1 are overlapping. It has been demonstrated as described in Seiffertet al, J.Biol. Chem. 269: 2659-2666 (1994) that high affinityvitronectin binding to active PAI-1 is primarily via the somatomedin Bdomain. The inventors tested whether vitronectin and recombinantsomatomedin B domain would also inhibit uPAR binding to vitronectin, andfound that both molecules inhibit, whereas a point mutation of thedomain abolishes their inhibition.

[0068] We identified bacteriophage peptides that are homologous to thesomatomedin B domain of vitronectin, the binding site of PAI-1. Thesomatomedin B domain of vitronectin blocks uPAR binding, and accordinglywe examined the sequences of bacteriophage derived peptides 7 and 18 forhomology to this domain. As shown in FIG. 4, there is a conserved motif,LXXArY (where X is a hydrophilic residue, and Ar=F,Y) between residues24-28 of the somatomedin B domain and clone 7 and 18 peptides. Inaddition, clones 7 and 18 share the sequence E-L-D just N-terminal tothe conserved leucine, whereas the related sequence D-E-L is found inthe somatomedin B domain of vitronectin at residues 22-24, adjacent tothe conserved sequence LCSYY.

[0069] To determine which residues in peptide 7 are important for uPARbinding and inhibition of vitronectin binding, we replaced each residueseparately with alanine, and tested the resulting peptides forinhibition of bacteriophage binding to uPAR, and blockade of the bindingof uPA1-48:uPAR complexes to vitronectin. The results shown in FIG. 5,indicate that the residues conserved between the peptides andvitronectin are important for activity in these assays.

[0070] Further, we determined that recombinant uPAR domain2-3 fragmentbinds bacteriophage but not uPA1-48. uPAR is the only member of theLy6/CD59 family to contain three repeats of the homologous cysteinecontaining domain Plough et al, FEBS Lett. 349: 163-168 (1994). Ourprevious work suggests that the binding site for vitronectin on uPAR isin domains 2 and 3 (D23) as described in Wei et al, J.Biol.Chem. 269:32380-32388 (1994). To further address this question the inventorsexpressed in baculovirus infected Sf9 insect cells a fragment of suPAR,residues 93-313, predicted to encompass the second and third CD59homologous domains with a C-terminal 6 amino acid epitope tag. Thesecreted protein was purified on an anti-epitope affinity column, andwas tested first for its ability to compete in the suPAR binding assay.There was no competition in this assay at 100 nM D23, in contrast tointact suPAR which shows an IC50 of 0.1 nM under the same conditions.

[0071] The inventors then tested the ability of various uPARbacteriophage displayed ligands to bind to immobilized D23. The resultsshown in FIG. 6, indicate that the ligands fall into three differentclasses with respect to binding to D23 and sUPAR. Clone 20 and 13-32bind signficantly only to intact sUPAR, whereas clones 9 and 25 bindequivalently to the D23 fragment and full-length receptor. Bacteriophagebearing clones 7 and 18 peptides show an intermediate degree of bindingto D23, and substantially better binding to an intact receptor.

[0072] Integrins are a class of heterodimeric receptors implicated inadhesive interactions that regulate cell trafficking and intracellularsignalling events important to cellular differentiation, migration andsurvival as described in Dustin et al, Nature 329: 846 (1987) andShattil et al, Curr. Opin. Cell Biol. 6: 695 (1994). Adhesion of cellsvia integrins requires, in addition to ligand binding, a reorganizationof intregrin distribution and assembly of connecting elements that linkintegrins to the cytoskeleton as described in Miyamoto et al, Science267:883 (1995) and Burridge et al, Annu. Rev. Cell Biol. 4: 487 (1988).β1 integrins have been extensively studied in this regard. Thecytoplasmic tail of β1 chains binds talin and alpha-actinin, whichthemselves interact directly with actin as described in Otey et al, J.Cell. Biol. 111: 721 (1990), and Schaller et al, J. Cell. Biol. 130:1181 (1988). Further, the assembly of such cytoskeletal connections isnot strictly a consequence of cell surface expression, but frequentlyrequires secondary cell signaling as described in Faull et al, J. Cell.Biol. 121: 155 (1993), Masumoto et al, J. Biol. Chem. 268: 228 (1993),and Burn et al, Proc. Nat'l. Acad. Sci. U.S.A. 85: 497 (1988). Beforethe experimental events that gave rise to the present invention,integrin-associated proteins which might mediate dynamic alterations inthe functional state of integrins remained largely undefined.

[0073] We determined that expression of uPAR not only confersadhesiveness for vitronectin but markedly diminishes β1-dependentadhesion of embryonic kidney cells (293 cells) to fibronectin. The studywas based on an observation that expression of uPAR in 293 cells alteredtheir integrin-dependent fibronectin and collagen adhesiveness. A phagedisplay peptide library was screened for uPAR-binding phages. A numberof uPAR-binding peptides as described in Goodson et al, Proc. Nat'lAcad. Sci. U.S.A. 91: 7129 (1994). Peptide 25 and several controls wassynthesized, purified, and screened for their effect on adhesion.Peptide 25, but not the controls was found to abrogateglycophosphatidyl-inositol (GPI) linked uPAR dependent adhesion of 293cells to vitronectin with an IC₅₀ of about 60 μM. Peptide 25 but not thecontrols, largely disrupted the β1/caveolin/uPAR complexes atconcentrations which blocked adhesion, about 100 μM. These observationsindentify a previously unrecognized functional unit within the cellmembrane that regulates cellular adhesiveness. This unit consists of aGPI-anchored receptor (uPAR), an integrin, and caveolin, and likelyother proteins known to associate with the cytoplasmic faces of β1integrins and caveolin, including cytoskeletal elements.

[0074] To explore whether uPAR binds to integrins, nontransfected 293cells were allowed to adhere to fibronectin or collagen in the presenceof recombinant soluble uPAR (suPAR). The results indicated that suPARinhibited adhesion of fibronectin and collagen in a dose dependentmanner, and the inhibitor effect was reversible with the addition of a100 μM peptide 25, but not a control. It was concluded that uPARinteracts with integrins that are in an active conformation and in sodoing markedly altered integrin function. It was also shown that peptide25 (100 μM) abrogated the interaction between another integrin, Mac-1and uPAR, in the U937 cell line.

[0075] Studies to determine the functional consequences of uPAR/integrininteractions on cellular migration were also conducted, with the resultthat altered cell migration was observed in the presence of uPAR bycreating a loss of integrin-dependent adhesiveness. Loss of stablecellular adhesion has been linked to malignant transformation, tumorcell invasion, and metastasis in several experimental and clinicalsituations as described in Huttenlocher et al, Cell Biol. 7: 697 (1995),Burchill et al, BioEssays 16: 225 (1994), and Lukashev et al, J. Biol.Chem. 26: 18311 (1994).

[0076] The invention includes the development of reagents such as thatprototyped by peptide 25 demonstrated to disupt uPAR/integrinassociations and restore integrin function, or reagents comparable tosoluble uPAR which impair integrin function, such as for example,antibodies to the site on integrin of uPAR:integrin binding, for use inmodifying inflammation and tumor progression.

[0077] The sequences selected in this study which bind to suPAR, asrepresented by peptides 7 and 25, have distinct binding sites, based onseveral lines of evidence. First, these peptides show different effectson anilino-8-napthalenesulfonate (ANS) fluorescence and as competitorsfor uPA1-48 binding as depicted in the table in FIG. 6. Second, onlypeptide 7 inhibits complex binding to vitronectin. Third, bacteriophage25 shows equivalent binding to D23 and suPAR, whereas 7 shows about50-fold reduced binding to D23. Peptide 18 appears to be of the sameligand family as 7, since it shows significant homology at the sequencelevel, and the conserved residues are important for clone 7 binding asindicated in FIG. 5. In particular, all of the defined residues in themotifs ELD and LxxArY are functionally important as judged by alaninereplacement. In addition, peptide 18 blocks binding of complexes tovitronectin, as does peptide 7.

[0078] The invention also includes methods for screening for molecularmimics of the inhibitory activity of the peptides of the invention, forexample peptide 7 and peptide 25, for the purpose of identifying, forexample, small molecule or peptoid inhibitors of uPAR:vitronectin oruPAR:integrin binding interactions. Such antagonists of uPARinteractions can be, for example, peptide derivatives such as peptoids,small molecules, or polynucleotides. These antagonists are useful fordevelopment of therapeutics for treatment of conditions characterized byuPAR:vitronectin binding or by uPAR:integrin binding, or more generally,by upregulation of uPA and uPAR, where cell adhesion is compromised. Theinstant peptides and antagonist can be useful in treating a diseasestate or malady which is caused or exacerbated by the biologicalactivity of uPA or uPAR. The conditions may also be characterized, forexample, by cell migration and invasion, as seen in such disorders as,for example, tumor cell invasion, metastatic disease, and the conditionmay also be chronic inflammation.

[0079] Typically, the molecular mimics, peptoids or small molecules; oranalogs, variants, or derivatives of the instant peptides exhibit aK_(d) of less than 10 μM: more preferably, less than 5 μM, even morepreferably less than 1 μM: even more preferably less than 100 nM; evenmore preferably less than 10 nM. with huPAR or the complex ofhuPAR:integrin or vitronectin.

[0080] Any of the full-length, derivatives, or polypeptide or peptideinhibitors or antagonists of the invention can be cloned, expressed, orsynthesized by standard recombinant DNA or chemical techniques. Someexemplary expression systems that can be applied for these purposesfollow. Administration of the peptide, polypeptide, and polynucleotidetherapeutics of the invention can conducted by administration of thesynthesizied peptide or polypeptide, or by administration of apolynucleotide for expression in an animal, or by administration of anon-coding polynucleotide inhibitor. Further below are also providedmethods of making small molecule and peptoid library pools for screeningfor the desired activity. Also provided are gene therapy techniques foradministering a polynucleotide of the invention to a patient for thepurpose of expressing the polypeptide or peptide encoded by thepolynucleotide or nucleic acid molecule in the animal. In addition,non-coding nucleic acid molecules, such as for example, ribozymes andantisense molecules can be administered with an appropriatepharmaceutically acceptable carrier.

[0081] Expression Systems

[0082] Although the methodology described below is believed to containsufficient details to enable one skilled in the art to practice thepresent invention, other items not specifically exemplified, such asplasmids, can be constructed and purified using standard recombinant DNAtechniques described in, for example, Sambrook et al. (1989), MOLECULARCLONING, A LABORATORY MANUAL, 2d edition (Cold Spring Harbor Press, ColdSpring Harbor, N.Y.), and Ausubel et al., CURRENT PROTOCOLS IN MOLECULARBIOLOGY (1994), (Greene Publishing Associates and John Wiley & Sons, NewYork, N.Y.). under the current regulations described in United StatesDept. of HHS, NATIONAL INSTITUTE OF HEALTH (NLH) GUIDELINES FORRECOMBINANT DNA RESEARCH. These references include procedures for thefollowing standard methods: cloning procedures with plasmids,transformation of host cells, cell culture, plasmid DNA purification,phenol extraction of DNA, ethanol precipitation of DNA, agarose gelelectrophoresis, purification of DNA fragments from agarose gels, andrestriction endonuclease and other DNA-modifying enzyme reactions.

[0083] Expression in Bacterial Cells

[0084] Control elements for use in bacteria include promoters,optionally containing operator sequences, and ribosome binding sites.Useful promoters include sequences derived from sugar metabolizingenzymes, such as galactose, lactose (lac) and maltose. Additionalexamples include promoter sequences derived from biosynthetic enzymessuch as tryptophan (trp), the β-lactamase (bla) promoter system,bacteriophage λPL, and T7. In addition, synthetic promoters can be used,such as the tac promoter. The β-lactamase and lactose promoter systemsare described in Chang et al., Nature (1978) 275: 615, and Goeddel etal., Nature (1979) 281: 544; the alkaline phosphatase, tryptophan (trp)promoter system are described in Goeddel et al., Nucleic Acids Res.(1980) 8: 4057 and EP 36,776 and hybrid promoters such as the tacpromoter is described in U.S. Pat. No. 4,551,433 and de Boer et al.,Proc. Natl. Acad. Sci. USA (1983) 80: 21-25.

[0085] However, other known bacterial promoters useful for expression ofeukaryotic proteins are also suitable. A person skilled in the art wouldbe able to operably ligate such promoters to the coding sequences ofinterest, for example, as described in Siebenlist et al., Cell (1980)20: 269, using linkers or adaptors to supply any required restrictionsites. Promoters for use in bacterial systems also generally willcontain a Shine-Dalgarno (SD) sequence operably linked to the DNAencoding the target polypeptide. For prokaryotic host cells that do notrecognize and process the native target polypeptide signal sequence, thesignal sequence can be substituted by a prokaryotic signal sequenceselected, for example, from the group of the alkaline phosphatase,penicillinase, Ipp, or heat stable enterotoxin II leaders. The origin ofreplication from the plasmid pBR322 is suitable for most Gram-negativebacteria.

[0086] The foregoing systems are particularly compatible withEscherichia coli. However, numerous other systems for use in bacterialhosts including Gram-negative or Gram-positive organisms such asBacillus spp., Streptococcus spp., Streptomyces spp., Pseudomonasspecies such as P. aeruginosa, Salmonella typhimurium, or Serratiamarcescans, among others. Methods for introducing exogenous DNA intothese hosts typically include the use of CaCl₂ or other agents, such asdivalent cations and DMSO. DNA can also be introduced into bacterialcells by electroporation, nuclear injection, or protoplast fusion asdescribed generally in Sambrook et al. (1989), MOLECULAR CLONING: ALABORATORY MANUAL, 2d edition (Cold Spring Harbor Press, Cold SpringHarbor, N.Y.). These examples are illustrative rather than limiting.Preferably, the host cell should secrete minimal amounts of proteolyticenzymes. Alternatively, in vitro methods of cloning, e.g., PCR or othernucleic acid polymerase reactions, are suitable.

[0087] Prokaryotic cells used to produce the target polypeptide of thisinvention are cultured in suitable media, as described generally inSambrook et al. (1989), MOLECULAR CLONING: A LABORATORY MANUAL, 2dedition (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.).

[0088] Expression in Yeast Cells

[0089] Expression and transformation vectors, either extrachromosomalreplicons or integrating vectors, have been developed for transformationinto many yeasts. For example, expression vectors have been developedfor, among others, the following yeasts: Saccharomyces cerevisiae, asdescribed in Hinnen et al., Proc. Natl. Acad. Sci. USA (1978) 75: 1929;Ito et al., J. Bacteriol. (1983) 153: 163; Candida albicans as describedin Kurtz et al., Mol. Cell. Biol. (1986) 6: 142; Candida maltosa, asdescribed in Kunze et al., J. Basic Microbiol. (1985)25: 141; Hansenulapolymorpha, as described in Gleeson et al., J. Gen. Microbiol. (1986)132: 3459 and Roggenkamp et al., Mol. Gen. Genet. (1986) 202 :302);Kluyveromyces fragilis, as described in Das et al., J. Bacteriol. (1984)158: 1165; Kluyveromyces lactis, as described in De Louvencourt et al.,J. Bacteriol. (1983) 154: 737 and Van den Berg et al., Bio/Technotogy(1990) 8: 135; Pichia guillerimondii, as described in Kunze et al., J.Basic Microbiol. (1985) 25: 141; Pichia pastoris, as described in Cregget al., Mol. Cell. Biol. (1985) 5: 3376 and U.S. Pat. Nos. 4,837,148 and4,929,555; Schizosaccharomyces pombe, as described in Beach and Nurse,Nature (1981) 300: 706; and Yarrowia lipolytica, as described in Davidowet al., Curr. Genet. (1985) 10: 380 and Gaillardin et al., Curr. Genet.(1985) 10: 49, Aspergillus hosts such as A. nidulans, as described inBallance et al., Biochem. Biophys. Res. Commun. (1983) 112: 28289;Tilbum et al., Gene (1983) 26: 205-221 and Yelton et al., Proc. Natl.Acad. Sci. USA (1984) 81: 1470-1474, and A. niger, as described in Kellyand Hynes, EMBO J. (1985) 4: 475479; Trichoderma reesia, as described inEP 244,234, and filamentous fungi such as, e.g, Neurospora, Penicillium,Tolypocladium, as described in WO 91/00357.

[0090] Control sequences for yeast vectors are known and includepromoters regions from genes such as alcohol dehydrogenase (ADH), asdescribed in EP 284,044, enolase, glucokinase, glucose-6-phosphateisomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH),hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, and pyruvatekinase (PyK), as described in EP 329,203. The yeast PHO5 gene, encodingacid phosphatase, also provides useful promoter sequences, as describedin Myanohara et al., Proc. NatL Acad. Sci. USA (1983) 80: 1. Othersuitable promoter sequences for use with yeast hosts include thepromoters for 3-phosphoglycerate kinase, as described in Hitzeman etal., J. Biol. Chem. (1980) 255: 2073, or other glycolytic enzymes, suchas pyruvate decarboxylase, triosephosphate isomerase, and phosphoglucoseisomerase, as described in Hess et al., J. Adv. Enzyme Reg. (1968) 7:149 and Holland et al., Biochemistry (1978) 17:4900. Inducible yeastpromoters having the additional advantage of transcription controlled bygrowth conditions, include from the list above and others the promoterregions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism,metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization. Suitable vectors andpromoters for use in yeast expression are further described in Hitzeman,EP 073,657. Yeast enhancers also are advantageously used with yeastpromoters. In addition, synthetic promoters which do not occur in naturealso function as yeast promoters. For example, upstream activatingsequences (UAS) of one yeast promoter may be joined with thetranscription activation region of another yeast promoter, creating asynthetic hybrid promoter. Examples of such hybrid promoters include theADH regulatory sequence linked to the GAP trascription activationregion, as described in U.S. Pat. Nos. 4,876,197 and 4,880,734. Otherexamples of hybrid promoters include promoters which consist of theregulatory sequences of either the ADH2, GAL4, GAL10, or PHO5 genes,combined with the transcriptional activation region of a glycolyticenzyme gene such as GAP or PyK, as described in EP 164,556. Furthermore,a yeast promoter can include naturally occurring promoters of non-yeastorigin that have the ability to bind yeast RNA polymerase and initiatetranscription.

[0091] Other control elements which may be included in the yeastexpression vectors are terminators, for example, from GAPDH and from theenolase gene, as described in Holland et al., J. Biol. Chem. (1981) 256:1385, and leader sequences which encode signal sequences for secretion.DNA encoding suitable signal sequences can be derived from genes forsecreted yeast proteins, such as the yeast invertase gene as describedin EP 012,873 and JP 62,096,086 and the a-factor gene, as described inU.S. Pat. Nos. 4,588,684, 4,546,083 and 4,870,008; EP 324,274; and WO89/02463. Alternatively, leaders of non-yeast origin, such as aninterferon leader, also provide for secretion in yeast, as described inEP 060,057.

[0092] Methods of introducing exogenous DNA into yeast hosts are wellknown in the art, and typically include either the transformation ofspheroplasts or of intact yeast cells treated with alkali cations.

[0093] Transformations into yeast can be carried out according to themethod described in Van Solingen et al., J. Bact. (1977) 130:946 andHsiao et al., Proc. Natl. Acad. Sci. (USA) (1979) 76:3829. However,other methods for introducing DNA into cells such as by nuclearinjection, electroporation, or protoplast fusion may also be used asdescribed generally in Sambrook et al., cited above.

[0094] For yeast secretion the native target polypeptide signal sequencemay be substituted by the yeast invertase, α-factor, or acid5-phosphatase leaders. The origin of replication from the 2 μ plasmidorigin is suitable for yeast. A suitable selection gene for use in yeastis the trp1 genre present in the yeast plasmid described in Kingsman etal., Gene (1979) 7: 141 or Tschemper et al., Gene (1980) 10:157. Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan. Similarly, Leu2-deficientyeast strains (ATCC 20,622 or 38,626) are complemented by known plasmidsbearing the Leu2 Gene.

[0095] For intracellular production of the present polypeptides inyeast, a sequence encoding a yeast protein can be linked to a codingsequence of the polypeptide to produce a fusion protein that can becleaved intracellularly by the yeast cells upon expression. An example,of such a yeast leader sequence is the yeast ubiquitin gene.

[0096] Extression in Insect Cells

[0097] Baculovirus expression vectors (BEVs) are recombinant insectviruses in which the coding sequence for a foreign gene to be expressedis inserted behind a baculovirus promoter in place of a viral gene,e.g., polyhedrin, as described in Smith and Summers, U.S. Pat. No.,4,745,051.

[0098] An expression construct herein includes a DNA vector useful as anintermediate for the infection or transformation of an insect cellsystem the vector generally containing DNA coding-for a baculovirustranscriptional promoter, optionally but preferably, followed downstreamby an insect signal DNA sequence capable of directing secretion of adesired protein, and a site for insertion of the foreign gene encodingthe foreign protein, the signal DNA sequence and the foreign gene beingplaced under the transcriptional control of a baculovirus promoter, theforeign gene herein being the coding sequence of the polypeptide.

[0099] The promoter for use herein can be a baculovirus transcriptionalpromoter region derived from any of the over 500 baculoviruses generallyinfecting insects, such as, for example, the Orders Lepidoptera,Diptera, Orthoptera, Coleoptera and Hymenoptera including, for example,but not limited to the viral DNAs of Autographo californica MNPV, Bombyxmori NPV, rrichoplusia ni MNPV, Rachlplusia ou MNPV or Galleriamellonella MNPV. Thus, the baculovirus transcriptional promoter can be,for example, a baculovirus immediate-early gene IEI or IEN promoter, animmediate-early gene in combination with a baculovirus delayed-earlygene promoter region selected from the group consisting of a 39K and aHindIII fragment containing a delayed early gene; or a baculovirus lategene promoter. The immediate-early or delayed ery promoters can beenhanced with transcriptional enhancer elements.

[0100] Particularly suitable for use herein is the strong polyhedrinpromoter of the baculovirus, which directs a high level of expression ofa DNA insert, as described in Friesen et al. (1986) “The Regulation ofBaculovirus Gene Expression” in: THE MOLECULAR BIOLOGY OF BACULOVIRUSES(W.Doerfler, ed.): EP 127,839 and EP 155,476; and the promoter from thegene encoding the p10 protein, as described in Vlak et al., J. Gen.Virol. (1988) 69:765-776.

[0101] The plasmid for use herein usually also contains the polyhedrinpolyadenylation signal, as described in Miller et al., Ann. Rev.Microbiol. (1988) 42:177 and a procaryotic ampicillin-resistance (amp)gene and an origin of replication for selection and propagation in E.coli. DNA encoding suitable signal sequences can also be included and isgenerally derived from genes for secreted insect or baculovirusproteins, such as the baculovirus polyhedrin gene, as described inCarbonell et al., Gene (1988) 73:409, as well as mammalian signalsequences such as those derived from genes encoding human a-interferonas described in Maeda et al., Nature (1985) 315:592-594; humangastrin-releasing peptide, as described in Lebacq-Verheyden et al., Mol.Cell. Biol. (1988) 8: 3129; human IL-2, as described in Smith et al.,Proc. Natl. Acad. Sci. USA (1985) 82:8404; mouse IL-3, as described inMiyajima et al., Gene (1987) 58:273; and human glucocerebrosidase, asdescribed in Martin et al., DNA (1988) 7:99.

[0102] Numerous baculoviral strains and variants and correspondingpermissive insect host cells from hosts such as Spodoptera frugiperda(caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito),Drosophila melanogaster (fruitfly), and Bombyx mori host cells have beenidentified and can be used herein. See, for example, the description inLuckow et al., Bio/Technology(1988) 6: 47-55, Miller et al., in GENETICENGINEERING (Setlow, J.K. et al. eds.), Vol. 8 (Plenum Publishing,1986), pp. 277-279, and Maeda et al., Nature, (1985) 315: 592-594. Avariety of such viral strans are publicly available, e.g., the L-1variant of Autographa californica NPV and the Bm-5 strain of Bombyx moriNPV. Such viruses may be used as the virus for transfection of hostcells such as Spodoptera frugiperda cells.

[0103] Other baculovirus genes in addition to the polyhedrin promotermay be employed to advantage in a baculovirus expression system. Theseinclude immediate-early (alpha), delayed-early (beta), late (gamma), orvery late (delta), according to the phase of the viral infection duringwhich they are expressed. The expression of these genes occurssequentially, probably as the result of a “cascade” mechanism oftranscriptional regulation. Thus, the immediate-early genes areexpressed immediately after infection, in the absence of other viralfunctions, and one or more of the resulting gene products inducestranscription of the delayed-early genes. Some delayed-early geneproducts, in turn, induce transcription of late genes, and finally, thevery late genes are expressed under the control of previously expressedgene products from one or more of the earlier classes. One relativelywell defined component of this regulatory cascade is IEI, a preferredimmediate-early gene of Autographo californica nuclear polyhedrosisvirus (AcMNPV). IEI is pressed in the absence of other viral functionsand encodes a product that stimulates the transcription of several genesof the delayed-early class, including the preferred 39K gene, asdescribed in Guarino and Summers, J. Virol. (1986) 57:563-571 and J.Virol. (1987) 61:2091-2099 as well as late genes, as described in Guannoand Summers, Virol. (1988) 162:4441 51.

[0104] Immediate-early genes as described above can be used incombination with a baculovirus gene promoter region of the delayed-earlycategory. Unlike the immediate-early genes, such delayed-early genesrequire the presence of other viral genes or gene products such as thoseof the immediate-early genes. The combination of immmediate-early genescan be made with any of several delayed-early gene promoter regions suchas 39K or one of the delayed-early gene promoters found on the HindIIIfragment of the baculovirus genome. In the present instance, the 39 Kpromoter region can be linked to the foreign gene to be expressed suchthat expression can be further controlled by the presence of IEI, asdescribed in L. A. Guarino and Summers (1986a), cited above; Guarino &Summers (1986b) J. Virol., (1986) 60:215-223, and Guarino et al.(1986c), J. Virol. (1986) 60:224-229.

[0105] Additionally, when a combination of immediate-early genes with adelayed-early gene promoter region is used, enhancement of theexpression of heterologous genes can be realized by the presence of anenhancer sequence in direct cis linkage with the delayed-early genepromoter region. Such enhancer sequences are characterized by theirenhancement of delayed-early gene expression in situations where theimmediate-early gene or its product is limited. For example, the hr5enhancer sequence can be linked directly, in cis, to the delayed-earlygene promoter region, 39K, thereby enhancing the expression of thecloned heterologous DNA as described in Guarino and Summers (1986a),(1986b), and Guarino et al. (1986).

[0106] The polyhedrin gene is classified as a very late gene. Therefore,trnscription from the polyhedrin promoter requires the previousexpression of an unknown, but probably large number of other viral andcellular gene products. Because of this delayed expression of thepolyhedrin promoter, state-of-the-art BEVs, such as the exemplary BEVsystem described by Smith and Summers in, for example, U.S. Pat. No.,4,745,051 will express foreign genes only as a result of gene expressionfrom the rest of the viral genome, and only after the viral infection iswell underway. This represents a limitation to the use of existing BEVs.The ability of the host cell to process newly synthesized proteinsdecreases as the baculovirus infection progresses. Thus, gene expressionfrom the polyhedrin promoter occurs at a time when the host cell'sability to process newly synthesized proteins is potentially diminishedfor certain proteins such as human tissue plasminogen activator. As aconsequence, the expression of secretory glycoproteins in BEV systems iscomplicated due to incomplete secretion of the cloned gene product,thereby trapping the cloned gene product within the cell in anincompletely processed form.

[0107] While it has been recognized that an insect signal sequence canbe used to express a foreign protein that can be cleaved to produce amature protein, the present invention is preferably practiced with amammalian signal sequence appropriate for the gene expressed.

[0108] An exemplary insect signal sequence suitable herein is thesequence encoding for a Lepidopteran adipokinetic hormone (AKH) peptide.The AKH family consists of short blocked neuropeptides that regulateenergy substrate mobilization and metabolism in insects. In a preferredembodiment, a DNA sequence coding for a Lepidopteran Manduca sexta AKHsignal peptide can be used. Other insect AKH signal peptides, such asthose from the Orthoptera Schistocerca gregaria locus can also beemployed to advantage. Another exemplary insect signal sequence is thesequence coding for Drosophila cuticle proteins such as CPI, CP2, CP3 orCP4.

[0109] Currently, the most commonly used transfer vector that can beused herein for introducing foreign genes into AcNPV is pAc373. Manyother vectors, known to those of skill in the art, can also be usedherein. Materials and methods for baculovirus/insect cell expressionsystems are commercially available in a kit form from companies such asInvitrogen (San Diego Calif.) (“MaxBac” kit). The techniques utilizedherein are generally known to those skilled in the art and are fullydescribed in Summers and Smith, A MANUAL OF METHODS FOR BACULOVIRUSVECTORS AND INSECT CELL CULTURE PROCEDURES, Texas AgriculturalExperiment Station Bulletin No. 1555, Texas A&M University (1987); Smithet al. Mol. Cell. Biol. (1983) 3: 2156, and Luckow and Summers (1989).These include, for example, the use of pVL985 which alters thepolyhedrin start codon from ATG to ATT, and which introduces a BamHIcloning site 32 basepairs downstream from the ATT, as described inLuckow and Summers, Virology (1989) 17:31.

[0110] Thus, for example, for insect cell expression of the presentpolypeptides, the desired DNA sequence can be inserted into the transfervector, using known techniques. An insect cell host can be cotransformedwith the transfer vector containing the inserted desired DNA togetherwith the genomic DNA of wild type baculovirus, usually bycotransfection. The vector and viral genome are allowed to recombineresulting in a recombinant virus that can be easily identified andpurified. The packaged recombinant virus can be used to infect insecthost cells to express a desired polypeptide.

[0111] Other methods that are applicable herein are the standard methodsof insect cell culture, cotransfection and preparation of plasmids areset forth in Summers and Smith (1987), cited above. This reference alsopertains to the standard methods of cloning genes into AcMNPV transfervectors, plasmid DNA isolation, transferring genes into the AcmMNPVgenome, viral DNA purification, radiolabeling recombinant proteins andpreparation of insect cell culture media. The procedure for thecultivation of viruses and cells are described in Volkman and Summers,J. Virol. (1975) 19:820-832 and Volkian, al., J. Virol. (1976)19:820-832.

[0112] Expression in Mammalian Cells

[0113] Typical promoters for mammalian cell expression of thepolypeptides of the invention include the SV40 early promoter, the CMVpromoter, the mouse mammary tumor virus LTR promoter, the adenovirusmajor late promoter (Ad MLP), and the herpes simplex virus promoter,among others. Other non-viral promoters, such as a promoter derived fromthe murine metallothionein gene, will also find use in mammalianconstructs. Mammalian expression may be either constitutive or regulated(inducible), depending on the promoter. Typically, transcriptiontermination and polyadenylation sequences will also be present, located3′ to the translation stop codon. Preferably, a sequence foroptimization of initiation of translation, located 5′ to the polypeptidecoding sequence, is also present Examples of transcriptionterminator/polyadenylation signals include those derived from SV40, asdescribed in Sambrook et al. (1989), cited previously. Introns,containing splice donor and acceptor sites, may also be designed intothe constructs of the present invention.

[0114] Enhancer elements can also be used herein to increase expressionlevels of the mammalian constructs. Examples include the SV40 early geneenhancer, as described in Dijkema et al, EMBO J. (1985) 4:761 and theenhancer/promoter derived from the long terminal repeat (LTR) of theRous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad.Sci. USA (1982b) 79:6777 and human cytomegalovirus, as described inBoshart et al., Cell (1985) 41:521. A leader sequence can also bepresent which includes a sequence encoding a signal peptide, to providefor the secretion of the foreign protein in mammalian cells. Preferably,there are processing sites encoded between the leader fragment and thegene of interest such that the leader sequence can be cleaved either invivo or in vitro. The adenovirus tripartite leader is an example of aleader sequence that provides for secretion of a foreign protein inmammalian cells.

[0115] Once complete, the mammalian expression vectors can be used totransform any of several mammalian cells. Methods for introduction ofheterologous polynucleotides into mammalian cells are known in the artand include dextran-mediated transfection, calcium phosphateprecipitation, polybrene mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) in liposomes,and direct microinjection of the DNA into nuclei. General aspects ofmammalian cell host system transformations have been described by Axelin U.S. Pat. No. 4,399,216.

[0116] The mammalian host cells used as responsive cells or producingcells in the invention may be cultured in a variety of mediaCommercially available media such as Ham's F10 (Sigma), MinimalEssential Medium ([MEM], Sigma), RPMI-1640 (Sigma), and Dulbecco'sModified Eagle's Medium ([DMEM], Sigma) are suitable for culturing thehost cells. In addition, any of the media described in Ham and Wallace,Meth. Enz. (1979) 58:44, Barnes and Sato, Anal. Biochem. (1980) 102:255,U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, or 4,560,655, WO90/103430, WO 87/00195. and U.S. RE 30,985, may be used as culture mediafor the host cells. Any of these media may be supplemented as necessaryto create optimal conditions for the function of the cells according tothe method of the invention, including supplementation as necessary withhormones and/or other growth factors such as insulin, transferrin, orepidermal growth factor, salts (such as sodium chloride, calcium,magnesium, and phosphate), buffers (such as HEPES), nucleosides (such asadenosine and thymidine), antibiotics (such as Gentamycin™M drug), traceelements (defined as inorganic compounds usually present at finalconcentrations in the micromolar range), and glucose or an equivalentenergy source range). Any other necessary supplements may also beincluded at appropriate concentrations that would be known to thoseskilled in the art. The culture conditions, such as temperature, pH, andthe like, are those previously used with the host cell selected forexpression, and will be apparent to the ordinarily skilled artisan.

[0117] Gene therapy strategies for delivery of constructs of theinvention can utilize viral or non-viral vector approaches in in vivo orex vivo modality. Expression of such coding sequence can be inducedusing endogenous mammalian or heterologous promoters. Expression of thecoding sequence in vivo can be either constitutive or regulated.

[0118] For delivery using viral vectors, any of a number of viralvectors can be used, as described in Jolly, Cancer Gene Therapy 1: 51-64(1994). For example, the coding sequence can be inserted into plasmidsdesigned for expression in retroviral vectors, as described in Kimura etal., Human Gene Therapy (1994) 5: 845-852, adenoviral vectors, asdescribed in Connelly et al., Human Gene Therapy (1995) 6: 185-193,adeno-associated viral vectors, as described in Kaplitt et al., NatureGenetics (1994) 6: 148-153 and sindbis vectors. Promoters that aresuitable for use with these vectors include the Moloney retroviral LTR,CMV promoter and the mouse albumin promoter. Replication incompetentfree virus can be produced and injected directly into the animal orhumans or by transduction of an autologous cell ex vivo, followed byinjection in vivo as described in Zatioukal et al., Proc. Natl. Acad.Sci. USA (1994) 91: 5148-5152.

[0119] The altered coding sequence can also be inserted into plasmid forexpression of the uPAR polypeptide in vivo or ex vivo. For in vivotherapy, the coding sequence can be delivered by direct injection intotissue or by intravenous infusion. Promoters suitable for use in thismanner include endogenous and heterologous promoters such as CMV.Further, a synthetic T7T7OB promoter can be constructed in accordancewith Chen et al. (1994), Nucleic Acids Res. 22: 2114-2120, where the T7polymerase is under the regulatory control of its own promoter anddrives the transcription of the uPAR coding sequence, which is alsoplaced under the control of a T7 promoter. The coding sequence can beinjected in a formulation comprising a buffer that can stablize thecoding sequence and facilitate transduction thereof into cells and/orprovide targeting, as described in Zhu et al., Science (1993) 261:209-211.

[0120] Expression of the coding sequence in vivo upon delivery for genetherapy purposes by either viral or non-viral vectors can be regulatedfor maximal efficacy and safety by use of regulated gene expressionpromoters as described in Gossen et al., Proc. Natl. Acad. Sci. USA(1992) 89:5547-5551. For example, the uPAR coding sequence can beregulated by tetracycline responsive promoters. These promoters can beregulated in a positive or negative fashion by treatment with theregulator molecule.

[0121] For non-viral delivery of the coding sequence, the sequence canbe inserted into conventional vectors that contain conventional controlsequences for high level expression, and then be incubated withsynthetic gene transfer molecules such as polymeric DNA-binding cationslike polylysine, protamnine, and albumin, linked to cell targetingligands such as asialoorosomucoid, as described in Wu and Wu, J. Biol.Chem. (1987) 262: 4429-4432; insulin, as described in Hucked et al.,Biochem. Pharmacol. 40: 253-263 (1990); galactose, as described in Planket al., Bioconjugate Chem. 3:533-539 (1992); lactose, as described inMidoux et al., Nucleic Acids Res. 21: 871-878 (1993); or transferrin, asdescribed in Wagner et al., Proc. Natl. Acad. Sci. USA 87:3410-3414(1990). Other delivery systems include the use of liposomes toencapsulate DNA comprising the uPAR gene under the control of a varietyof tissue-specific or ubiquitously-active promoters, as described inNabel et al., Proc. Natl. Acad. Sci. USA 90: 11307-11311 (1993), andPhilip et al., Mol. Cell Biol. 14: 2411-2418 (1994). Further non-viraldelivery suitable for use includes mechanical delivery systems such asthe biolistic approach, as described in Woffendin et al., Proc. Natl.Acad. Sci. USA (1994) 91(24): 11581-11585. Moreover, the uPAR codingsequence and the product of expression of such can be delivered throughdeposition of photopolymerized hydrogel materials. Other conventionalmethods for gene delivery that can be used for delivery of the uPARcoding sequence include, for example, use of hand held gene transferparticle gun, as described in U.S. Pat. No. 5,149,655; use of ionizingradiation for activating transferred gene, as described in U.S. Pat. No.5,206,152 and PCT application WO92/11033.

[0122] Application of gene therapy technology with regard to thepeptides and polypeptides of the invention and their analogues orvariants can be made in disease states where, for example, activity ofany of uPAR is detrimental to the patien. It is also conceived by theinventors that gene therapy using the polypeptides and peptides of theinvention and their analogues or variants is appropriate when treatingconditions of cytoskeletal disruption, for example, in vivo expressionof antagonists or dominant negatives to interupt, for example, theuPAR:integrin binding pair formation and the cellular responses, such ascellular migration, that result from the binding pair formation of uPARand integrin.

[0123] In general, gene therapy can be applied according to theinvention in all situations where uPAR forms a binding pair interactionwith vitronectin or integrin and acts to modulate cytoskeletal integrityand affect cellular migration, by administering according to a genetherapy protocol, of a sufficient amount of a peptide of the inventionor its analogue, variant, or dominant negative, for example, formodulating the normal activity of uPAR binding pair interactions.

[0124] Applications of the peptides of the invention, whetheradministered by a gene therapy protocol, or otherwise, can be made inthe context of treatment of a patient afflicted by a conditioncharacterized by cytoskeletal disruption and/or also including cellularmigration. Conditions of cancer and/or inflammatory conditions areexamples of such conditions.

[0125] For the purpose of the invention, based on the sequence andfunction of the novel peptides herein, assays can be developed forscreening small molecule library pools for functional uPAR:vitronectinand uPAR:integrin inhibitors, antagonists, and agonists for use incontrolling, for example, cytoskeletal disruption and cellularmigration. These inhibitors, antagonists, or agonists can beadministered to the animal, and can be administered with apharmaceutically acceptable carrier, including, for example, liposomescompositions such as Depofoam™, and other carriers such as, for example,Focalgel™.

[0126] Small molecule libraries may be used to screen for the ability ofthe small molecule to mimic, synergize or attenuate any action of SIP,and can be made as follows. A “library” of peptides may be synthesizedand used following the methods disclosed in U.S. Pat. No. 5,010,175,(the '175 patent) and in PCT WO91/17823. In method of the '175 patent, asuitable peptide synthesis support, for example, a resin, is coupled toa mixture of appropriately protected, activated amino acids.

[0127] The method described in WO91/17823 is similar. However, insteadof reacting the synthesis resin with a mixture of activated amino acids,the resin is divided into twenty equal portions, or into a number ofportions corresponding to the number of different amino acids to beadded in that step, and each amino acid is coupled individually to itsportion of resin. The resin portions are then combined, mixed, and againdivided into a number of equal portions for reaction with the secondamino acid. Additionally, one may maintain separate “subpools” bytreating portions in parallel, rather than combining all resins at eachstep. This simplifies the process of determining which peptides areresponsible for any observed alteration of gene expression in aresponsive cell.

[0128] The methods described in WO91/17823 and U.S. Pat. No. 5,194,392enable the preparation of such pools and subpools by automatedtechniques in parallel, such that all synthesis and resynthesis may beperformed in a matter of days.

[0129] A further alternative agents include small molecules, includingpeptide analogs and derivatives, that can act as stimulators orinhibitors of gene expression, or as ligands or antagonists. Somegeneral means contemplated for the production of peptides, analogs orderivatives are outlined in CHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS,PEPTIDES, AND PROTEINS—A SURVEY OF RECENT DEVELOPMENTS, Weinstein, B.ed., Marcell Dekker, Inc., publ. New York (1983). Moreover, substitutionof D-amino acids for the normal L-stereoisomer can be carried out toincrease the half-life of the molecule.

[0130] Peptoids, polymers comprised of monomer units of at least somesubstituted amino acids, can act as small molecule stimulators orinhibitors herein and can be synthesized as described in PCT 91/19735.Presently preferred amino acid substitutes are N-alkylated derivativesof glycine, which are easily synthesized and incorporated intopolypeptide chains. However, any monomer units which allow for thesequence specific synthesis of pools of diverse molecules areappropriate for use in producing peptoid molecules. The benefits ofthese molecules for the purpose of the invention is that they occupydifferent conformational space than a peptide and as such are moreresistant to the action of proteases.

[0131] Peptoids are easily synthesized by standard chemical methods. Thepreferred method of synthesis is the “submonomer” technique described byR. Zuckermann et al., J. Am. Chem. Soc. (1992) 114:10646-7. Synthesis bysolid phase techniques of heterocyclic organic compounds in whichN-substituted glycine monomer units forms a backbone is described incopending application entitled “Synthesis of N-Substituted Oligomers”filed on Jun. 7, 1995 and is herein incorporated by reference in full.Combinatorial libraries of mixtures of such heterocyclic organiccompounds can then be assayed for the ability to alter gene expression.

[0132] Synthesis by solid phase of other heterocyclic organic compoundsin combinatorial libraries is also described in copending applicationU.S. Ser. No. 08/485,006 entitled “Combinatorial Libraries ofSubstrate-Bound Cyclic Organic Compounds” filed on Jun. 7, 1995, hereinincorporated by reference in full. Highly substituted cyclic structurescan be synthesized on a solid support by combining the submonomer methodwith powerful solution phase chemistry. Cyclic compounds containing one,two, three or more fused rings are formed by the submonomer method byfirst synthesizing a linear backbone followed by subsequentintramolecular or intermolecular cyclization as described in the sameapplication.

[0133] Suitable carriers for the therapeutics of the invention foradministration in a patient, including but not limited to moleculescapable of antagonizing the inhibitory effects of the peptides of theimvention (for example peptides 7, 9, 18, and 25 and analogs or variantsof these), including, for example small molecules, peptides, peptoids,polynucleotides and polypeptides, may be large, slowly metabolizedmacromolecules such as proteins, polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids, amino acid copolymers, andinactive virus particles. Such carriers are well known to those ofordinary skill in the art. Pharmaceutically acceptable salts can be usedtherein, for example, mineral acid salts such as hydrochlorides,hydrobromides, phosphates, sulfates, and the like; and the salts oforganic acids such as acetates, propionates, malonates, benzoates, andthe like. A thorough discussion of pharmaceutically acceptableexcipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (MackPub. Co., N.J. 1991). Pharmaceutically acceptable carriers intherapeutic compositions may contain liquids such as water, saline,glycerol and ethanol. Additionally, auxiliary substances, such aswetting or emulsifying agents, pH buffering substances, and the like,may be present in such vehicles. Typically, the therapeutic compositionsare prepared as injectables, either as liquid solutions or suspensions;solid forms suitable for solution in, or suspension in, liquid vehiclesprior to injection may also be prepared. Liposomes are included withinthe definition of a pharmaceutically acceptable carrier. The term“liposomes” refers to, for example, the liposome compositions describedin U.S. Pat. No. 5,422,120, WO 95/13796, WO 94/23697, WO 91/14445 and EP524,968 B1. Liposomes may be pharmaceutical carriers for the peptides,polypeptides or polynucleotides of the invention, or for combination ofthese therapeutics.

[0134] Further objects, features, and advantages of the presentinvention will become apparent from the following detailed description.It should be understood, however, that the detailed description, whileindicating preferred embodiments of the present invention, is given byway of illustration only, since various changes and modifications withinthe spirit and scope of the invention will become apparent to thoseskilled in the art from this detailed description. The invention is alsonot limited to any theories of action of the elements of the invention.

EXAMPLE 1 Affinity Selection of 15 mer Random Peptide Library onuPAR:uPA1-48 Complexes

[0135] Soluble recombinant human urokinase receptor (suPAR) wasexpressed and secreted from baculovirus-infected Sf9 insect cells, asdescribed in Goodson et al, Proc.Natl.Acad.Sci. USA 91: 7129-7133(1994). The EGF-like domain of human urokinase (uPA residues 1-48) wasexpressed from recombinant yeast as described Stratton-Thomas et al,Prot.Eng. 8: 463-470 (1995). UPA1-48 was purified by a revision of thepublished procedure, involving ion exchange chromatography and reversephase HPLC under reducing conditions, followed by a refolding step andrechromatography on reversed phase HPLC of the oxidized material.Soluble uPAR was purified on a column of immobilized uPA1-48, eluted atlow pH, biotinylated according to Kaufman et al, Anal.Biochem. 211:261-266 (1993) and purified on a Soft-Avidin column (PromegaCorporation, Madison, Wis.). The uPAR fragment encompassing domains 2and 3 (amino acids 93-313) with a C-termninal epitope tag of E-Y-M-P-M-Eas described in Grussenmeyer et al, Proc.Natl.Acad.Sci. USA 82:7952-7954 (1985) was expressed in baculovirus infected Sf9 insect cells,and purified from the conditioned media by affinity chromatography on ananti-epitope antibody column. Peptides were synthesized at ChironMimotopes (Melbourne, Australia) with free amino termini and amidatedcarboxyl termini, and were greater than 70% pure by HPLC and MSanalysis. A variant of clone 20 peptide was prepared with the sequence:AE PMPHSLNFSQYAWYT (SEQ ID NO 7). A scrambled version of clone 7 had thesequence: VEYRDAYSYPQYLSYLE (SEQ ID NO 8). Recombinant PAI-1 wasobtained from American Diagnostica. Horse-radish peroxidase (HRP)conjugated streptavidin was from Pierce Chemical, Rockford, Ill.Anti-M13 antibody was from Pharmacia, Piscataway, N.J.

[0136] Affinity selections were performed on streptavidin coatedmagnetic beads (Dynal, Rochester, N.Y.) Biotinylated suPAR (1.5 μg) wasmixed with 3.5 μg of uPA1-48 in a total volume of 100 μl for 30 minutesat room temperature. Magnetic beads were blocked with PBS/1% BSA(PSB/BSA) for 30 minutes and then suPAR:uPA1-48 complexes were added inPBS/0.1% BSA, and incubated at room temperature for 2 hours. Beads werethen washed 3 times with PBS/BSA and resuspended with an aliquot of the15 mer random peptide library in 500 μl. For comparison an identicalaliquot of the beads was incubated with the parent bacteriophage vector(LP67, as described in Devlin et al, Science 249: 404 406 (1990).Binding of bacteriophage was for 45 minutes at room temperature followedby 7 washes with 2 ml PBS/BSA and elution of bound bacteriophage with500 μl 60 mM glycine, 1.5 M urea, pH 2.5. Eluted bacteriophage weretitered and amplified as described in Goodson et al, Proc.Natl.Acad.Sci.USA 91: 7129-7133 (1994), and Devlin et al, Science 249: 404-406 (1990).Amplified bacteriophage were then selected for additional rounds onsuPAR:uPA1-48 complexes as described above. DNA sequencing was performedon PCR amplified inserts from individual bacteriophage plaques by thedideoxy method.

EXAMPLE 2 Bacteriophaae Binding to sUPAR

[0137] Streptavidin, 100 μl (0.1 mg/ml) in 50 mM Na₂CO₃, pH 9.6, wasadded to MaxiSorp wells (Nunc), incubated overnight at 4 C, and thenwashed with PBS/BSA. Biotinylated sUPAR (25 nM in PBS/BSA) was added tothe wells and incubated for 2 hours at room temperature prior towashing. Competitive peptide inhibitors were added to the wellsimmediately prior to addition of the bacteriophage. The wells wereincubated for one hour at room temperature, then washed and boundbacteriophage eluted with 6M urea in 0.1N HCl, pH 2.5. After 15 minutes,the urea eluate was brought to neutral pH by addition of 2M Tris baseand the bacteriophage titers of input stocks and elutions measured byplaque formation assay. Results were expressed as the percent of inputbacteriophage which bind to the wells. Alternatively, the amount ofbacteriophage was determined in an ELISA where phage were preincubatedwith HRP-conjugated anti-M13 antibody for 30 minutes at room temperaturebefore dispensing into wells prepared as above and incubated for onehour at room temperature. The final anti-M13 conjugate dilution was1:4000. After washing, TMB substrate (100 μl/well) was added and colordevelopment was stopped with 0.8N H₂SO₄ (100 μl/well). The absorbance at450 nm was then measured in a 96 well plate reader.

[0138] Novel peptide sequences are obtained by panning uPA1-48:uPARcomplexes. Selection of high affinity peptide ligands for the uPAbinding site on uPAR was a relatively efficient process as described inGoodson et al, Proc.Natl.Acad.Sci.USA 91: 7129-7133 (1994), was extendedby selecting for peptide-displaying bacteriophage with affinity foradditional, functionally important sites on uPAR by including an excessof recombinant EGF-like domain of uPA (uPA1-48) to reduce selection ofuPA binding site peptides as described in Stratton-Thomas et al,Prot.Eng. 8: 463470 (1995). The EGF-like domain is the receptor bindingmotif as described in Appella et al, J.Biol.Chem. 262: 4437-4440 (1987)and Robbiati et al Fibrinol. 4: 53-60 (1990), and binds to uPAR withsimilar affinity (0.1-5 nM) as uPA as described in Mazar et al,Fibrinol. 6: 49-55 (1992). The 15 mer random peptide bacteriophagelibrary as descrobed in Devlin et al, Science 249: 404-406 (1990) wasaffinity selected on suPAR:uPA1-48 complexes immobilized on magneticbeads. The yield of bacteriophage increased 30 fold, from 0.008% atround 2 to 0.24% at round 3 suggesting enrichment for bindingbacteriophage.

[0139] Twenty-eight independent bacteriophage were isolated and therandom peptide encoding DNA segments sequenced. From these 28bacteriophage, 23 different sequences were obtained, but only fourclones (7, 9, 18, and 25) had substantial yields (>2%), whenindividually affinity selected on immobilized sUPAR. This is in contrastto previous results of affinity selection on uPAR alone, where themajority of selected bacteriophage bound with substantial yield asdescribed in Goodson et al, Proc.Natl.Acad.Sci. USA 91: 7129-7133(1994). The yields of these four bacteriophage were determined on suPARin the presence and absence of uPA1-48. In addition, the encodedpeptides were synthesized and tested as competitors in a suPAR bindingassay as described in Stratton-Thomas et al, Prot.Eng. 8: 463-470(1995), and Kaufman et al Anal.Biochem. 211: 261-266 (1993). Theseresults are summarized in the Table in FIG. 6.

[0140] The binding of the selected bacteriophage was largely unaffectedby the presence of a 1000 fold molar excess of uPA1-48. In contrast, thepreviously described bacteriophage (clone 20 and uPA13-32) which bind tothe uPA binding site, both gave yields of 2-5% on suPAR but were reducedto background levels (greater than 500-fold reduction) in the presenceof uPA1-48, as reported in Goodson et al, Proc.Natl.Acad.Sci. USA 91:7129-7133 (1994). These results suggest that the bacteriophage selectedon uPA1-48: uPAR complexes represent distinct classes of uPAR ligandsfrom clone 20 and uPA13-32.

[0141] It was also shown by the inventors that bacteriophage bound tosUPAR domain 2-3 fragment. Protein G, 100 μl, 1 mg/ml in 50 mM Na₂CO₃,pH 9.6, was added to MaxiSorp wells, incubated overnight at 4° C. andthen washed with PBS/BSA. Fifty μl of monoclonal antibody to the epitopetag EYMPME was added at 1 mg/ml in PBS/BSA and incubated for 2 hours atroom temperature. The wells were washed, recombinant sUPAR domain 2-3(1.7 μM in PBS/BSA) was added and incubated for 1.5 hours at roomtemperature. The wells were washed prior to the addition ofbacteriophage (approximately 10⁸ pfu), and then treated as described inthe previous section for binding to suPAR.

EXAMPLE 3 Vitronectin Binding Assay

[0142] Vitronectin was purified from human plasma by the method ofYatohgo et al, Cello Struct.and Funct. 13: 281-292 (1988). Purifiedvitronectin was diluted to 20 μg/ml in PBS containing 1 mM CaCl₂ and 0.5mM MgCl₂, dispensed at 50 μl/well into Immulon II wells (Dynatech,Chantilly, Va.), incubated overnight at 4° C. and washed with PBS/BSA.Biotinylated sUPAR was diluted to 20 nM in PBS/BSA, incubated with orwithout test ligand for 30 minutes at room temperature (22° C.),dispensed at 100 μl/well and incubated for 90 minutes. Wells were thenwashed and horseradish peroxidase (HRP)-conjugated streptavidin wasadded at 0.4 μg/ml in PBS/2% BSA for 1 hour followed by washing andaddition of 100 μl/well TMB substrate. The color development was stoppedwith 100 μl of 0.8N H₂SO₄ and the absorbance at 450 nm measured in a 96well plate reader (Dynatech, Chantilly, Va.). Antagonistic effects oftest ligands were measured as described above except the ligands wereincubated with 20 nM biotinylated suPAR in the presence of 20 nMuPA1-48.

[0143] It was found that uPA1-48:uPAR complexes bind with high affinityto vitronectin. In order to analyze the effects of the various peptideligands on the uPAR:vitronectin interaction, we developed an in vitroELISA based assay for this interaction, in which biotinylated suPAR anduPA1-48 bind to immobilized, urea purified vitronectin, and the boundsUPAR is detected with HRP conjugated streptavidin. Under the conditionsof the assay binding of biotinylated uPAR to vitronectin is strictlydependent on uPA1-48, as shown in FIG. 1. The apparently stoichiometricbinding of the uPA1-48:suPAR complexes to vitronectin indicates that theaffinity of this interaction is higher than the concentration of complex(Kd<20 nM).

[0144] It was also found that bacteriophage derived peptides blockcomplex binding and cell adhesion to vitronectin. The ability of thevarious bacteriophage derived peptides to affect binding of uPA1-48:uPARcomplexes to vitronectin was assessed in the ELISA assay. Two classes ofpeptides were effective antagonists in this assay. First, clone 20 anduPA13-32, which compete directly for uPA1-48 binding to sUPAR, reducedbinding. An analog of clone 20 peptide, which shows greatly reducedreceptor binding activity did not affect binding to vitronectin. Second,clones 7 and 18, which show greatly reduced competition for uPA1-48binding (see Table in FIG. 6) also inhibit complex binding, while ascrambled version of clone 7 did not. None of the peptides when testedalone increased the binding of biotinylated suPAR to vitronectin. Athird peptide, clone 25, which bound efficiently to suPAR as abacteriophage, had little or no effect on uPA1-48 stimulated vitronectinbinding. In order to test whether the clone 7 and 18 peptides bounddirectly at the vitronectin binding site on uPAR, and inhibitedvitronectin binding by uPAR:uPA1-48 by direct competition for that site,the inventors examined the effects of vitronectin on the binding ofthese bacteriophage. Vitronectin reduced bacteriophage binding to theuPA1-48:suPAR complex by 5-10 fold, consistent with the hypothesis thatthese peptides mimic vitronectin as a uPAR ligand.

[0145] Previous results had shown that vitronectin binding by uPARcorrelated with cell adhesion of stimulated U937 cells as described inWei et al, J.Biol.Chem. 269: 32380-32388 (1994). It was found also thatclone 7 peptide could block uPAR mediated adhesion of these cells,whereas the scrambled version of the same peptide had no effect.

[0146] Additionally, binding of uPA1-48:uPAR complexes to vitronectinwas shown to be blocked by PAI-1, vitronectin, and the somatomedin Bdomain of vitronectin. Another function of vitronectin has beendetermined to be stabilization of the active conformation of PAI-1,which appears to occur via the somatomedin B domain of vitronectin, asdescribed in Seiffert et al J.Biol.Chem. 269: 2659-2666 (1994). PAI-1 isa very efficient competitor of uPA1-48:suPAR complexes binding tovitronectin, with an apparent IC50 of 10 nM. This suggested to theinventors that the binding site of uPAR and PAI-1 are overlapping. Ithas been demonstrated previously that high affinity vitronectin bindingto active PAI-1 is primarily via the somatomedin B domain, as describedin Seiffert et al J.Biol.Chem. 269: 2659-2666 (1994). Thus, theinventors tested whether vitronectin and recombinant somatomedin Bdomain would also inhibit uPAR binding to vitronectin. Accordingly, theinventors showed that molecules inhibit, whereas a point mutation of thedomain does not.

[0147] It was also then determined that the bacteriophage peptides arehomologous to the somatomedin B domain of vitronectin, which is also thebinding site of PAI-1. The sequences of bacteriophage derived peptides 7and 18 were examined for homology to this domain. As shown in FIG. 3,there is a conserved motif, LXXArY (where X is a hydrophilic residue,and Ar=F,Y) between residues 24-28 of the somatomedin B domain and clone7 and 18 peptides. In addition, clones 7 and 18 share the sequence E-L-Djust N-terminal to the conserved leucine, whereas the related sequenceD-E-L is found in the somatomedin B domain of vitronectin at residues22-24, adjacent to the conserved sequence LCSYY.

[0148] To determine which residues in peptide 7 are important for uPARbinding and inhibition of vitronectin binding, we replaced each residueseparately with alanine, and tested the resulting peptides forinhibition of bacteriophage binding to uPAR, and blockade of the bindingof uPA1-48:uPAR complexes to vitronectin. The results shown in FIG. 4,indicate that the residues conserved between the peptides andvitronectin are important for activity in these assays.

EXAMPLE 4 SuPAR:1-Anilino-8-Napthalenesulfonate (ANS) FluorescenceMeasurements

[0149] Determination of the effect of various peptide ligands onsUPAR/ANS fluorescence was performed following a procedure similar tothat of Ploug et al, Biochem. 33: 8991-8997 (1994). Fluorescenceemission spectra of sUPAR/ANS solutions with or without competitors wereobtained using an Hitachi F-4500 fluorescence spectrophotometer with anexcitation wavelength of 386 nm, 5-nm band-pass excitation and emissionslits, and a 10 mm path length quartz cuvette. The emission spectra from400 to 600 nm were recorded. For competition measurements, dilutions ofa stock sUPAR/ANS solution were made to give individual 0.5 ml aliquotswith a final concentration of 2 μM sUPAR, 10 μM ANS, and 0 to 20 μMcompetitor in PBS containing 10% DMSO. Fluorescence measurements weremade after a one hour incubation at 25° C.

[0150] It was found that ANS fluorescence enhancement distinguished thepeptide sequences To further analyze the binding sites of these peptideligands the inventors examined their effects on the fluorescenceenhancement of ANS which occurs upon uPAR binding, and which has beenshown to correlate with occupancy of the uPA binding site and thefunctional state of the uPAR molecule of Ploug et al, Biochem. 33:8991-8997 (1994). The effects of several peptide uPAR ligands on ANSfluorescence enhancement in the presence of uPAR had the expected resultthat uPA1-48 and clone 20 reduce ANS fluorescence, consistent with theirpotent activity in the receptor binding assay. Clone 7 also reducedfluorescence in a dose dependent manner, although at higherconcentrations, while clone 25 peptide has no effect at up to 20 μM.These results suggested that clone 7, 20, and uPA1-48 share some commonbinding determinants or a common binding conformation of uPAR with ANS,whereas clone 25 binds to a distinct site.

EXAMPLE 5 Recombinant UPAR Domain2-3 Fragment Binds Bacteriophage butnot uPA1-48

[0151] UPAR is the only member of the Ly6/CD59 family to contain threerepeats of the homologous cysteine containing domain as described inPlough et al, FEBS Lett. 349: 163-168 (1994). Previous work by theinventors suggested that the binding site for vitronectin on uPAR is indomains 2 and 3 (D23) as described by Wei et al, J.Biol. Chem. 269:32380-32388 (1994). To further address this question we expressed inbaculovirus infected Sf9 insect cells a fragment of suPAR, residues93-313, predicted to encompass the second and third CD59 homologousdomains with a C-terninal 6 amino acid epitope tag. The secreted proteinwas purified on an anti-epitdpe affinity column, and was tested firstfor its ability to compete in the suPAR binding assay. There was nocompetition in this assay at 100 nM D23, in contrast to intact suPARwhich shows an IC50 of 0.1 nM under the same conditions.

[0152] The inventors then tested the ability of various uPARbacteriophage displayed ligands to bind to immobilized D23. The resultsshown in FIG. 5, indicate that the ligands fall into three differentclasses with respect to binding to D23 and sUPAR. Clone 20 and 13-32bind signficantly only to intact suPAR, whereas clones 9 and 25 bindequivalently to the D23 fragment and full-length receptor. Bacteriophagebearing clones 7 and 18 peptides show an intermediate degree of bindingto D23, and substantially better binding to intact receptor.

EXAMPLE 6 Identification of uPAR:Integrin Binding and Binding Site

[0153] In order to ascertain whether cytoskeletal connecting elementsimportant to integrin-dependent adhesion were also involved in adhesionmediated by uPAR, embryonic kidney cells (293 cells) were engineered tocoexpress uPAR along with a chimeric protein comprised of the β1cytoplasmic tail fused with the transmembrane domain of complementaritydetermining region 4 (CD4). Expression of this chimeric β1 construct haspreviously been shown to exert a dominant negative effect onintegrin-mediated adhesion by sequestering cytoplasmic elements whichbind P chains as described in Lukashev et al, J. Biol. Chem. 26: 18311(1994). Co-expression of uPAR with β1 cytoplasmic domains completelyabrogated uPAR-dependent vitronectin adhesion. Clones expressing fulllength or truncated β1 cytoplasmic tails prepared as in Lukashev et al,J. Biol. Chem. 26: 18311 (1994) were transfected with cDNA for GPI-uPARand selected as in Wei et al, J. Biol. Chem. 169: 32380 (1994). Chimericβ1 expression was induced by cadmium for 6 hours prior to assayingadhesion to vitronectin at 37° C. Following induction of full length β1chimerics, essentially no cells were adherent to vitronectin-coatedsurfaces whereas co-transfectants expressing the truncated β1 adheredavidly.

[0154] Cells co-expressing uPAR with a control, truncated β1 cytoplasmicdomain unable to connect with cytoskeletal proteins as described inLukashev et al J. Biol. Chem. 26: 18311 (1994) adhered normally.Inhibition of adhesion at 37° C. developed despite comparable urokinaseand vitronectin binding at 4° C. among the co-transfectants, suggestingcompetition between β1 cytoplasmic tails and uPAR for cytoskeletalconnecting elements important to adhesion.

[0155] Based on these results, immunoprecipitation experiments wereconducted to determine whether uPAR was physically associated withnative β1 integrins. A stable transfectant expressing a chimeric uPARcomprised of the extracellular domain of uPAR fused with the IL-2R alphatransmembrane domain and short cytoplasmic tail (TM-uPAR) was generatedas a control. This chimeric uPAR binds urokinase comparably to GPI-uPARas described in Hui et al, J. Biol. Chem. 269:8153 (1994) The fulllength cDNA for the human urokinase receptor and human interleukin-2receptor were isolated from human macrophages and human T cells,respectively, by reverse transcription and polymerase chain reaction. Achimeric cDNA construct encoding the extracellular domains of the uPAR(amino acids 1-281) and the transmembrane/cytoplasmic domams of IL-2Ralhpa (amino acids 218-251) was prepared. The chimeric cDNA wassubcloned into pBluescript, verified by nucleotide sequenceing(Sequenase, United States Biochemical Corp) then digested with XbaI andXhoI and finally subcloned into the pCEP4 expression vector.Co-transfectants were shown to bind equivalent amounts of vitronectinand urokinase at 4° C. by methods as described in Wei et al, J. Biol.Chem. 269: 32380(1994).

[0156] Immunoblotting confirmed comparable expression of GPI-uPAR andTM-uPAR in 293 cells as well as comparable urokinase and vitronectinbinding. When triton X 100 (0.2%) insoluble fraction of GPI-uPAR 293cells is solubilized in polar detergents, immunoprecipitation of β1clearly co-precipitates uPAR as described in Filardo et al, J. Cell.Biol. 1995, in press: Cells (5×10⁶) were cultured overnight, washedtwice with microtubule stabilization buffer (0.1M PIPES, pH 6.9, 2Mglycerol, 1 mM EDTA, and 1 mM magnesium acetate), and then extracted onice for 5 minutes in buffer containing 0.2% Triton X 100 and inhibitors(1 mM sodium orthovandate, 1 mM phenylsulfonyl fluoride, 10 mg/mlleupeptin). The insoluble residues were solubolized at 4° C. for 20minutes in 1× RIPA buffer (150 mM sodium chloride, 50 mM Tris-HCl, pH7.5, 1% deoxycholate, 0.1% sodium dodecyl sulfate, 1% Triton X-100)supplemented with protease inhibitors. The triton soluble fraction wasdiluted 1:1 with 2× RIPA buffer. Both fractions were centrifuged for 10minutes at 6000 rpm, and then precleared by incubation with nonimmuneserum and protein A-agarose for 2 hours at 4° C.

[0157] Supernatants were transferred to fresh tubes and incubated withantibodies against β1 or caveolin for 2 hours at 4° C. Immune complexeswere recovered with protein A-agarose. The washed immunoprecipitateswere subjected to 8% SDS-PAGE, and transferred onto a nitrocellulosemembrane. The filters were blocked in 5% nonfat dried milk, and probedwith anti-uPAR Mab R2 (from E. Ronne, Finsen Lab, Denmark), 1 μg/ml. Theblots were washed and incubated with HRP conjugated antibodies for onehour. After washing, the membranes were developed using enhancedchemiluminescence (NEN Du Pont, Wilmington, Del.) according to themanufacturer's protocol.

[0158] A similar result was obtained when a rat monoclonal β1 antibodywas substituted. β1 immuno-precipitations of the triton X 100 detergentsoluble fraction revealed no uPAR. In addition, much less or noassociation of uPAR with β1 could be demonstrated with TM-uPAR in eithertriton fraction.

[0159] Cell adhesion assays were conducted to determin whether theobserved uPAR/β1/caveolin complexes were functionally relevant. Althoughboth GPI-uPAR and TM-uPAR bound vitronectin comparably at 4° C., onlyGPI-uPAR expressing cells showed enhanced adhesion to vitronectin,suggesting that the association of uPAR with β1 is necessary.

[0160] To test this hypothesis further, a phage display peptide librarywas screened for uPAR-binding phages. A number of phage peptides wereisolated as described in Goodson et al, Proc. Natl. Acad. Sci. U.S.A.91: 7129 (1994). One phage displayed a uPAR-binding peptide whichneither blocked urokinase/uPAR or vitronectin/uPAR associations. Thispeptide, peptide 25 and several controls were synthesized, purified, andscreened for their effect on adhesion. Peptide 25, but not the controls,was found to abrogate GPI-uPAR dependent adhesion of 293 cells tovitronectin, IC₅₀ of about 60 μM. Peptide 25 had no effect on adhesionto fibronectin by nontransfected 293 cells. Immunoprecipitationexperiments were then conducted to assess the effect of this and controlpeptides on the association of uPAR with β1. Peptide 25, but not acontrol peptide, largely disrupted the β1/caveolin/uPAR complexes atconcentrations which blocked adhesion (100 μM). Several additionalnon-inhibitory peptides from the original screening were tested andfound to have no effect on β1/uPAR co-precipitation, confirming that theβ1/GPI-uPAR/caveolin complexes operate as an adhesive unit.

[0161] In addition, minimal motifs for peptide 25 were determined by analanine scan of peptide 25, looking for binding to uPAR: residue changes% inhibition to alanine of phage binding NONE (clone 25) 100 S-1 99 T-269 Y-3 21 H-4 17 H-5 100 L-6 0 S-7 99 L-8 96 G-9 16 Y-10 16 M-11 35 Y-1217 T-13 39 L-14 98 N-15 100

[0162] These data suggest that the minimal motif necessary forinhibition of binding is YHXLXXGYMYT (SEQ ID NO 5) in clone 25 where Xis any amino acid.

[0163] These data indicate that uPAR associates with and modifiesfunction of certain integrins. This association both promotes adhesionto a migration toward a specific matrix protein, vitronectin, anddestabilizes the normal adhesive function of integrins. In vivo, theability of uPAR to destabilize integrin-dependent attachments isreinforced by the concurrent binding of the protease urokinase.

EXAMPLE 7 Identification of Additional Ligands that Bind to uPAR

[0164] In a uPAR binding assay, the following analogs were tested in acompetition with phage displaying either peptide 25 or peptide 9. Theanalogs comprise both natural and unnatural amino acids.

[0165] In the table below, the analog sequences are listed with theamino terminus of the analogs printed on the left. The analog sequencesutilized the one letter amino acid abbreviations unless otherwise noted.The lower case letters indicate a D-amino acids. for example “s”indicates a D-serine. Analogs 2-4 and 31-96 have a free amino terminusand a C-terminal carboxamides. Analogs 5-30 comprise an acetylatedterminus (Ac-oligomer-NH₂).

[0166] The analogs were tested in an assay utilizing soluble uPAR,similar to the method described in Example 2. The analogs were testedfor their ability to compete with phage displaying either peptide 9 orpeptide 25. Analogs 3-61 were tested in competition with peptide 25.Analogs 62-96 were tested in competition with peptide 9. Approximately10⁸ plaque forming units of the phage were used in the assay. # Sequence# Sequence 49 AEStYHHLSLGYMYTLN 50 AESTyHHLSLGYMYTLN 3 AESTYHHLSLGYMYTLN51 AESTYhHLSLGYMYTLN 4 AESTYHHLSLGYMYTLN 52 AESTYHhLSLGYMYTLN 5AESTYHHLSLGYMYTLN 53 AESTYHHlSLGYMYTLN 6 AESTYHHLSLGYMYTL 54AESTYHHLsLGYMYTLN 7 AESTYHHLSLGYMYT 55 AESTYHHLSlGYMYTLN 8AESTYHHLSLGYMY 56 AESTYHHLSLGyMYTLN 9 AESTYHHLSLGYM 57 AESTYHHLSLGYmYTLN10 AESTYHHLSLGY 58 AESTYHHLSLGYMyTLN 11 AESTYHHLSLG 59 AESTYHHLSLGYMYtLN12 AESTYHHLSL 60 AESTYHHLSLGYMYTlN 13 AESTYHHLS 61 AESTYHHLSLGYMYTLn 14AESTYHHL 62 AEFFKLGPNGYVYLHSA 15 ESTYHHLSLGYMYTLN 63 AEFFKLGPNGYVYLHSA16 STYHHLSLGYMYTLN 64 AEFFKLGPNGYVYLHSA 17 TYHHLSLGYMYTLN 65AAFFKLGPNGYVYLHSA 18 YHHLSLGYMYTLN 66 AEAFKLGPNGYVYLHSA 19 HHLSLGYMYTLN67 AEFAKLGPNGYVYLHSA 20 HLSLGYMYTLN 68 AEFFALGPNGYVYLHSA 21 LSLGYMYTLN69 AEFFKAGPNGYVYLHSA 22 SLGYMYTLN 70 AEFFKLAPNGYVYLHSA 23 LGYMYTLN 71AEFFKLGANGYVYLHSA 24 AESTYHHLSLG 72 AEFFKLGPAGYVYLHSA 25 ESTYHHLSLGY 73AEFFKLGPNAYVYLHSA 26 STYHHLSLGYM 74 AEFFKLGPNGAVYLHSA 27 TYHHLSLGYMY 75AEFFKLGPNGYAYLHSA 28 YHHLSLGYMYT 76 AEFFKLGPNGYVALHSA 29 HHLSLGYMYTL 77AEFFKLGPNGYVYAHSA 30 HLSLGYMYTLN 78 AEFFKLGPNGYVYLASA 31AESTYHHGPNGYMYTLN 79 AEFFKLGPNGYVYLHAA 32 AESTYHHsPNGYMYTLN 80AEFFKLsPNGYVYLHSA 33 AESTYHHaPNGYMYTLN 81 AEFFKLaPNGYVYLHSA 34AESTFHHLSLGYMYTLN 82 aEFFKLGPNGYVYLHSA 35 AESTXHHLSLGYMYTLN 83AeFFKLGPNGYVYLHSA 36 AEST?HHLSLGYMYTLN 84 AEfFKLGPNGYVYLHSA 37AESTYHHLSLGFMYTLN 85 AEFfKLGPNGYVYLHSA 38 AESTYHHLSLGXMYTLN 86AEFFkLGPNGYVYLHSA 39 AESTYHHLSLG?MYTLN 87 AEFFKlGPNGYVYLHSA 40AESTYHHLSLGYMFTLN 88 AEFFKLGpNGYVYLHSA 41 AESTYHHLSLGYMXTLN 89AEFFKLGPnGYVYLHSA 42 AESTYHHLSLGYM?TLN 90 AEFFKLGPNGyVYLHSA 43AESTYHHLSLGYVYTLN 91 AEFFKLGPNGYvYLHSA 44 AESTYHHLSLGYJYTLN 92AEPFKLGPNGYVyLHSA 45 AESTYHHLSLGTbYTLN 93 AEFFKLGPNGYVYlHSA 46aESTYHHLSLGYMYTLN 94 AEFFKLGPNGYVYLhSA 47 AeSTYHHLSLGYMYTLN 95AEFFKLGPNGYVYLHsA 48 AEsTYHHLSLGYMYTLN 96 AEFFKLGPNGYVYLHSa

[0167] Results of Analogs 3-30

[0168] The analogs were tested at a concentration at 40 μM in the uPARcompetition assay with phage displaying peptide 25. The results belowshow which analogs were active. Active? Sequence @ 40 μM AES{dot over(T)}

H

SL

LN Y AESTYHHLSLGYMYTL Y AESTYHHLSLGYMYT Y AESTYHHLSLGYMY N AESTYHHLSLGYMN AESTYHHLSLGY N AESTYHHLSLG N AESTYHHLSL N AESTYHHLS N AESTYHHL N ESTYHHLSLGYMYTLN Y   STYHHLSLGYMYTLN Y    TYHHLSLGYMYTLN Y    YHHLSLGYMYTLN Y      HHLSLGYMYTLN N       HLSLGYMYTLN N       LSLGYMYTLN N         SLGYMYTLN N          LGYMYTLN N AESTYHHLSLGN  ESTYHHLSLGY N   STYHHLSLGYM N    TYHHLSLGYMY N     YHHLSLGYMYT Y     HHLSLGYMYTL N       HHLSLGYMYTL N

[0169] Results of Analogs 31-61

[0170] Analogs 31-61 were tested for their ability to compete with phagedisplaying peptide 25. The active analogs were tested further at twoconcentrations, 5 μM and 2.5 μM. These concentrations were calculatedbased on the synthesis reactions. However, the sequences * were furthertested and determined to contain high amounts of amino acids and thequantity tested could have been higher than 5 μM or 2.5 μM.

[0171] The results of the testing with the active analogs are shownbelow: % Inhibition Sequence 5 uM 2.5 uM AES{dot over (T)}

H

SL

LN 59 45 AEST

HHLSLGYMYTLN 47 38 AEST

HHLSLGYMYTLN 94 70 AEST

HHLSLGYMYTLN 78 61 AESTYHHLSLGY

YTLN 82 63 AESTYHHLSLGY

YTLN 91 84* AESTYHHLSLGY

YTLN 59 31

ESTYHHLSLGYMYTLN 57 41 A

STYHHLSLGYMYTLN 61 35 AESTYHHL

LGYMYTLN 86 73 AESTYHHLS

GYMYTLN 80 56 AESTYHHLSLGYMYT

N 75 22 AESTYHHLSLGYMYTL

55 32

[0172] Results of Oligomers 62-79

[0173] An alanine scan was performed using the sequence of peptide 9.The sequences of analogs 62-79 are the same as peptide 9 except analanine residue was substituted at one position in the sequence. Analogs62-79 are all the possible alanine substitutions into peptide 9.

[0174] The results of the Ala scan show that alanine substitution forLeu at position 6, Tyr at position 11, Val at position 12, and Tyr atposition 13 destroyed receptor binding activity. Alanine substitution atfor Glu at position 2, Phe at position 4, or Leu at position 14decreased but did not destroy, the receptor binding activity of theoligomers as compared to peptide 9.

[0175] Results of Analogs 80-96

[0176] Analogs 80-96 were tested for their ability to compete with phagedisplaying peptide 9. The active analogs were tested further at twoconcentrations, 5 μM and 2.5 μM. These concentrations were calculatedbased on the synthesis reactions. However, the sequences * were furthertested and determined to contain low amounts of amino acids and thequantity tested could have been lower than 5 μM or 2.5 μM.

[0177] The results of the testing with the active analogs are shownbelow: % Inhibition Sequence 5 uM 2.5 uM A{dot over (E)}F{dot over (F)}K

GPNG

{dot over (L)}HSA 71 59 AEFFKLGPN

YVYLHSA 95 89 AEFFKLGPN

YVYLHSA 80 79

EFFKLGPNGYVYLHSA 41 24* A

FFKLGPNGYVYLHSA 59 44 AEFFKLG

NGYVYLHSA 76 80* AEFFKLGP

GYVYLHSA 13 15 AEFFKLGPNGYV

LHSA  3.3  8.5 AEFFKLGPNGYVYL

SA 41 36 AEFFKLGPNGYVYLH

A 59 47 AEFFKLGPNGYVYLHS

40 38*

[0178]

1 76 1 16 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide ligand 1 Ala Glu Pro Val Tyr Gln Tyr Glu Leu Asp SerTyr Leu Arg Ser Tyr 1 5 10 15 2 16 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic peptide ligand 2 Ala Glu Phe Phe LysLeu Gly Pro Asn Gly Tyr Val Tyr Leu His Ser 1 5 10 15 3 16 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptideligand 3 Ala Glu Leu Asp Leu Ser Thr Phe Tyr Asp Ile Gln Tyr Leu Leu Arg1 5 10 15 4 16 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide ligand 4 Ala Glu Ser Thr Tyr His His Leu SerLeu Gly Tyr Met Tyr Thr Leu 1 5 10 15 5 11 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide ligand 5 Tyr HisXaa Leu Xaa Xaa Gly Tyr Met Tyr Thr 1 5 10 6 11 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide ligand 6 Phe LysLeu Xaa Xaa Xaa Gly Tyr Val Tyr Leu 1 5 10 7 16 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide ligand 7 Ala GluPro Met Pro His Ser Leu Asn Phe Ser Gln Tyr Ala Trp Tyr 1 5 10 15 8 16PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide ligand 8 Val Glu Tyr Arg Asp Ala Tyr Ser Tyr Pro Gln Tyr Leu SerTyr Leu 1 5 10 15 9 47 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide ligand 9 Asp Gln Glu Ser Cys Lys Gly Arg CysThr Glu Gly Phe Asn Val Asp 1 5 10 15 Lys Lys Cys Gln Cys Asp Glu LeuCys Ser Tyr Tyr Gln Ser Cys Cys 20 25 30 Thr Asp Tyr Thr Ala Glu Cys LysPro Gln Val Thr Arg Gly Asp 35 40 45 10 17 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide ligand 10 Ala GluPro Val Tyr Gln Tyr Glu Leu Asp Ser Tyr Leu Arg Ser Tyr 1 5 10 15 Tyr 1117 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide ligand 11 Ala Glu Leu Asp Leu Ser Thr Phe Tyr Asp Ile Gln TyrLeu Leu Arg 1 5 10 15 Thr 12 17 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide ligand 12 Ala Glu Pro Val Tyr GlnTyr Glu Leu Asp Ser Tyr Leu Arg Ser Tyr 1 5 10 15 Tyr 13 17 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptideligand 13 Ala Glu Phe Phe Lys Leu Gly Pro Asn Gly Tyr Val Tyr Leu HisSer 1 5 10 15 Ala 14 17 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide ligand 14 Ala Glu Leu Asp Leu SerThr Phe Tyr Asp Ile Gln Tyr Leu Leu Arg 1 5 10 15 Thr 15 17 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptideligand 15 Ala Glu Ser Thr Tyr His His Leu Ser Leu Gly Tyr Met Tyr ThrLeu 1 5 10 15 Asn 16 17 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide ligand 16 Ala Glu Pro Met Pro HisSer Leu Asn Phe Ser Gln Tyr Leu Trp Tyr 1 5 10 15 Thr 17 19 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptideligand 17 Cys Leu Asn Gly Gly Thr Ala Val Ser Asn Lys Tyr Phe Ser AsnLeu 1 5 10 15 His Trp Cys 18 6 PRT Artificial Sequence Description ofArtificial Sequence C-terminal epitope tag 18 Glu Tyr Met Pro Met Glu 15 19 15 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 19 Ala Glu Ser Thr Tyr His His Leu Ser Leu Gly Tyr MetTyr Thr 1 5 10 15 20 14 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 20 Ala Glu Ser Thr Tyr His His LeuSer Leu Gly Tyr Met Tyr 1 5 10 21 13 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic peptide 21 Ala Glu Ser Thr Tyr His HisLeu Ser Leu Gly Tyr Met 1 5 10 22 12 PRT Artificial Sequence Descriptionof Artificial Sequence Synthetic peptide 22 Ala Glu Ser Thr Tyr His HisLeu Ser Leu Gly Tyr 1 5 10 23 11 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 23 Ala Glu Ser Thr Tyr His His LeuSer Leu Gly 1 5 10 24 10 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 24 Ala Glu Ser Thr Tyr His His LeuSer Leu 1 5 10 25 9 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 25 Ala Glu Ser Thr Tyr His His Leu Ser 1 5 268 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 26 Ala Glu Ser Thr Tyr His His Leu 1 5 27 16 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 27 Glu SerThr Tyr His His Leu Ser Leu Gly Tyr Met Tyr Thr Leu Asn 1 5 10 15 28 15PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 28 Ser Thr Tyr His His Leu Ser Leu Gly Tyr Met Tyr Thr Leu Asn 15 10 15 29 14 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 29 Thr Tyr His His Leu Ser Leu Gly Tyr Met Tyr Thr LeuAsn 1 5 10 30 13 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 30 Tyr His His Leu Ser Leu Gly Tyr Met TyrThr Leu Asn 1 5 10 31 12 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 31 His His Leu Ser Leu Gly Tyr MetTyr Thr Leu Asn 1 5 10 32 11 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 32 His Leu Ser Leu Gly Tyr Met TyrThr Leu Asn 1 5 10 33 10 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 33 Leu Ser Leu Gly Tyr Met Tyr ThrLeu Asn 1 5 10 34 9 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 34 Ser Leu Gly Tyr Met Tyr Thr Leu Asn 1 5 358 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 35 Leu Gly Tyr Met Tyr Thr Leu Asn 1 5 36 11 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 36 Ala GluSer Thr Tyr His His Leu Ser Leu Gly 1 5 10 37 11 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 37 Glu Ser Thr TyrHis His Leu Ser Leu Gly Tyr 1 5 10 38 11 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 38 Ser Thr Tyr HisHis Leu Ser Leu Gly Tyr Met 1 5 10 39 11 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 39 Thr Tyr His HisLeu Ser Leu Gly Tyr Met Tyr 1 5 10 40 11 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 40 Tyr His His LeuSer Leu Gly Tyr Met Tyr Thr 1 5 10 41 11 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 41 His His Leu SerLeu Gly Tyr Met Tyr Thr Leu 1 5 10 42 11 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 42 His Leu Ser LeuGly Tyr Met Tyr Thr Leu Asn 1 5 10 43 17 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 43 Ala Glu Ser ThrTyr His His Gly Pro Asn Gly Tyr Met Tyr Thr Leu 1 5 10 15 Asn 44 17 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide44 Ala Glu Ser Thr Tyr His His Ser Pro Asn Gly Tyr Met Tyr Thr Leu 1 510 15 Asn 45 17 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 45 Ala Glu Ser Thr Tyr His His Ala Pro AsnGly Tyr Met Tyr Thr Leu 1 5 10 15 Asn 46 17 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 46 Ala Glu Ser ThrPhe His His Leu Ser Leu Gly Tyr Met Tyr Thr Leu 1 5 10 15 Asn 47 17 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide47 Ala Glu Ser Thr Xaa His His Leu Ser Leu Gly Tyr Met Tyr Thr Leu 1 510 15 Asn 48 17 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 48 Ala Glu Ser Thr Xaa His His Leu Ser LeuGly Tyr Met Tyr Thr Leu 1 5 10 15 Asn 49 17 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 49 Ala Glu Ser ThrTyr His His Leu Ser Leu Gly Phe Met Tyr Thr Leu 1 5 10 15 Asn 50 17 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide50 Ala Glu Ser Thr Tyr His His Leu Ser Leu Gly Xaa Met Tyr Thr Leu 1 510 15 Asn 51 17 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 51 Ala Glu Ser Thr Tyr His His Leu Ser LeuGly Xaa Met Tyr Thr Leu 1 5 10 15 Asn 52 17 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 52 Ala Glu Ser ThrTyr His His Leu Ser Leu Gly Tyr Met Phe Thr Leu 1 5 10 15 Asn 53 17 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide53 Ala Glu Ser Thr Tyr His His Leu Ser Leu Gly Tyr Met Xaa Thr Leu 1 510 15 Asn 54 17 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 54 Ala Glu Ser Thr Tyr His His Leu Ser LeuGly Tyr Met Xaa Thr Leu 1 5 10 15 Asn 55 17 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 55 Ala Glu Ser ThrTyr His His Leu Ser Leu Gly Tyr Val Tyr Thr Leu 1 5 10 15 Asn 56 17 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide56 Ala Glu Ser Thr Tyr His His Leu Ser Leu Gly Tyr Xaa Tyr Thr Leu 1 510 15 Asn 57 17 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 57 Ala Glu Ser Thr Tyr His His Leu Ser LeuGly Tyr Xaa Tyr Thr Leu 1 5 10 15 Asn 58 17 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 58 Ala Ala Phe PheLys Leu Gly Pro Asn Gly Tyr Val Tyr Leu His Ser 1 5 10 15 Ala 59 17 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide59 Ala Glu Ala Phe Lys Leu Gly Pro Asn Gly Tyr Val Tyr Leu His Ser 1 510 15 Ala 60 17 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 60 Ala Glu Phe Ala Lys Leu Gly Pro Asn GlyTyr Val Tyr Leu His Ser 1 5 10 15 Ala 61 17 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 61 Ala Glu Phe PheAla Leu Gly Pro Asn Gly Tyr Val Tyr Leu His Ser 1 5 10 15 Ala 62 17 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide62 Ala Glu Phe Phe Lys Ala Gly Pro Asn Gly Tyr Val Tyr Leu His Ser 1 510 15 Ala 63 17 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 63 Ala Glu Phe Phe Lys Leu Ala Pro Asn GlyTyr Val Tyr Leu His Ser 1 5 10 15 Ala 64 17 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 64 Ala Glu Phe PheLys Leu Gly Ala Asn Gly Tyr Val Tyr Leu His Ser 1 5 10 15 Ala 65 17 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide65 Ala Glu Phe Phe Lys Leu Gly Pro Ala Gly Tyr Val Tyr Leu His Ser 1 510 15 Ala 66 17 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 66 Ala Glu Phe Phe Lys Leu Gly Pro Asn AlaTyr Val Tyr Leu His Ser 1 5 10 15 Ala 67 17 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 67 Ala Glu Phe PheLys Leu Gly Pro Asn Gly Ala Val Tyr Leu His Ser 1 5 10 15 Ala 68 17 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide68 Ala Glu Phe Phe Lys Leu Gly Pro Asn Gly Tyr Ala Tyr Leu His Ser 1 510 15 Ala 69 17 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 69 Ala Glu Phe Phe Lys Leu Gly Pro Asn GlyTyr Val Ala Leu His Ser 1 5 10 15 Ala 70 17 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 70 Ala Glu Phe PheLys Leu Gly Pro Asn Gly Tyr Val Tyr Ala His Ser 1 5 10 15 Ala 71 17 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide71 Ala Glu Phe Phe Lys Leu Gly Pro Asn Gly Tyr Val Tyr Leu Ala Ser 1 510 15 Ala 72 17 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 72 Ala Glu Phe Phe Lys Leu Gly Pro Asn GlyTyr Val Tyr Leu His Ala 1 5 10 15 Ala 73 17 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 73 Ala Glu Phe PheLys Leu Ser Pro Asn Gly Tyr Val Tyr Leu His Ser 1 5 10 15 Ala 74 17 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide74 Ala Glu Phe Phe Lys Leu Ala Pro Asn Gly Tyr Val Tyr Leu His Ser 1 510 15 Ala 75 17 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 75 Ala Glu Phe Phe Lys Leu Gly Pro Asn SerTyr Val Tyr Leu His Ser 1 5 10 15 Ala 76 17 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 76 Ala Glu Phe PheLys Leu Gly Pro Asn Ala Tyr Val Tyr Leu His Ser 1 5 10 15 Ala

1. A method of identifying an orphan binding site on a targetpolypeptide sequence comprising the steps of, (a) providing a library ofpotential ligands, (b) providing the target polypeptide in contact witha known ligand for said target polypeptide, (c) contacting the targetpolypeptide and known ligand with the potential ligands, and (d)identifying the potential ligand that binds to said target polypeptidein the presence of the known ligand to form a binding pair with thetarget polypeptide.
 2. The method of claim 1, wherein said library ofpotential ligands comprises a bacteriophage library.
 3. The method ofclaim 1 wherein the target polypeptide sequence comprises a sequenceselected from the group consisting of a receptor, a ligand, a growthfactor, a polypeptide hormone, a cytokine, a differentiation factor, amolecule capable of signal transduction, an enzyme, and a polypeptideinvolved in extracellular matrix interactions.
 4. The method of claim 3wherein the receptor is a urokinase plasminogen activator receptor andthe known ligand comprises one selected from the group consisting ofvitronectin and uPA.
 5. The method of claim 1 wherein the potentialligands; comprise one selected from the group consisting of randomsynthesized peptides, small molecules, peptoids, polypeptides andpolynucleotides.
 6. The method of claim 1, wherein the potential ligandsare contacted with the target polypeptide and unknown ligand to identifya potential ligand that antagonizes a binding pair interaction betweenthe target polypeptide and an unknown ligand.
 7. The method of claim 6,wherein the unknown ligand is integrin.
 8. (Cancelled)
 9. (Cancelled)10. An isolated peptide that binds a urokinase plasminogen activatorreceptor (uPAR) and inhibits uPAR binding to vitronectin.
 11. Theisolated peptide of claim 10, comprising an amino acid sequence selectedfrom the group consisting of AEPVYQYELDSYLRSYY (SEQ ID NO: 1), andAELDLSTFYDIQYLLRT (SEQ ID NO:3).
 12. An isolated peptide that binds aurokinase plasminogen activator receptor (uPAR), comprising an aminoacid sequence selected from the group consisting of AEFFKLGPNGYVYLHSA(SEQ ID NO:2) and FKLXXXGYVYL (SEQ ID NOI where X is any amino acid. 13.(Cancelled)
 14. (Cancelled)
 15. (Cancelled)
 16. (Cancelled) 17.(Cancelled)
 18. A method of treating patient with a disordercharacterized by upregulation of uPA and uPAR comprising the steps of(a) providing an effective amount of an antagonist of a uPAR:integrinbinding pair, (b) administering the antagonist to the patient.
 19. Themethod of claim 18, wherein the antagonist is a peptide comprising anamino acid sequence selected from the group consisting ofAESTYHHLSLGYMYTLN (SEQ-ID NO:4), and YHXLXXGYMYT (SEQ ID NO:5), where Xis any amino acid.
 20. (Cancelled)
 21. The method of claim 18, whereinthe disorder characterized by upregulation of uPA and uPAR furthercomprises cellular migration.
 22. (Cancelled)
 23. (Cancelled) 24.(Cancelled)
 25. (Cancelled)
 26. (Cancelled)
 27. (Cancelled) 28.(Cancelled)
 29. (Cancelled)
 30. (Cancelled)
 31. (Cancelled)
 32. Apharmaceutical composition for treating a patient with a disordercharacterized by upregulation of uPA and uPAR comprising an effectiveamount of a nucleic acid encoding a peptide that comprises an amino acidsequence selected from the group consisting of AEPVYQYELDSYLRSYY (SEQ IDNO: 1), AEFFKLGPNGYVYLHSA (SEQ ID NO:2), AELDLSTFYDIQYLLRT (SEQ IDNO:3), AESTYHHLSLGYMYTLN (SEQ ID NOA), and YiIXLXXGYMYT (SEQ ID NO:5),and FKLXXXGYVYL (SEQ ID NO:6), where X is any amino acid.
 33. Thepharmaceutical composition of claim 32, wherein the pharmaceuticallyacceptable carrier comprises one selected from the group consisting of aliposome, a gel, a polymer matrix, a foam, and a buffer.